importance of gene therapy essay

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What Is Gene Therapy?

A New Type of Therapy for Genetic Diseases

• Why It's Done
• What to Expect

Frequently Asked Questions

Gene therapy is a type of treatment being developed to fight diseases that are caused by genetic defects. This is a relatively new medical intervention that is mainly in the experimental phase, including human trials and animal trials, for the treatment of some conditions, such as cystic fibrosis .

Gene therapy aims to change the unhealthy proteins that are produced as a result of disease-causing genes.

KATERYNA KON / SCIENCE PHOTO LIBRARY / Getty Images

What Is Gene Therapy? 

Some diseases are caused by a known genetic defect or a gene mutation. This means that there is a hereditary or acquired error in the DNA molecule that codes for producing a specific protein in the body. The altered protein doesn’t function as it should, resulting in a disease. 

The idea behind gene therapy is to direct the body to produce healthy proteins that do not cause disease.

This therapy involves the delivery of DNA or RNA. The RNA molecule is an intermediate molecule that is formed in the process of protein production. The genetic defect for some diseases has been identified, but many genetic mutations haven’t been identified (they might be in the future). 

Research is ongoing into ways to correct genetic defects that have been associated with certain diseases. There are different types and methods of gene therapy that are being investigated. 

Types of Gene Therapy 

Genetic mutations can be hereditary, which means that they are inherited from parents. Genetic defects can also be acquired, sometimes due to environmental factors, like smoking. 

Gene therapy is being evaluated as a potential treatment for both types of mutations. There are several ways of delivering the corrected DNA or RNA into a person’s body.

Most cells in your body are somatic cells. The only cells that are not somatic cells are the germline cells, which create the egg and sperm cells that can produce offspring.

Somatic gene therapy : Somatic gene therapy aims to correct a defect in the DNA of a somatic cell or to provide an RNA molecule to treat or prevent a genetic disease in the person who is undergoing the therapy. This treatment may be used if you have an inherited mutation or if the mutation developed due to environmental factors.

Germline gene therapy : Germline gene therapy aims to correct a defect in an egg or a sperm cell to prevent a hereditary disease from eventually affecting future offspring.

Bone Marrow 

Sometimes a person’s own cells can be removed from the bone marrow , genetically modified in a laboratory, and then reinserted into the body.

Viral Vector 

A viral vector is a virus that has been altered so that it will not cause a viral infection. It is then infused with the correct DNA or RNA sequence. The viral vector containing the correct gene may be injected into a person for delivery of gene therapy.

Stem Cells 

Stem cells are immature cells that have the potential to develop into different types of cells. Sometimes stem cells that have been genetically modified are transplanted into a person’s body to replace the defective cells as a way to treat disease. 

This technique uses a lipid (fat) to deliver the genetic DNA or RNA material. 

Why Is Gene Therapy Done? 

Some gene mutations direct the body to make disease-causing proteins. And some genetic mutations are not functional—they cause disease because the body lacks the healthy proteins that should be normally produced by the gene.

Gene therapy aims to direct the body to produce healthy proteins or to inhibit the production of defective proteins. This depends on the type of mutation that is causing the disease. 

Gene Augmentation Therapy: Replacing Mutated Genes

With gene augmentation, the goal is to help the body make a healthy protein.

Sometimes the DNA molecule can have a gene inserted into it. This is intended to permanently alter the DNA so that the body can make new cells with the correct DNA code. The new cells will then also make healthy products. 

Some research using gene augmentation therapy involves the insertion of a healthy DNA molecule or an RNA sequence into a cell, but not into the DNA of the recipient. This has been shown in experimental studies to trigger the production of healthy proteins, but future copies of the cell are not expected to contain the healthy gene. 

Gene Inhibition Therapy: Inactivating Mutated Genes 

Sometimes gene therapy aims to cancel out the activity of a mutated gene to prevent the production of a disease-causing protein. This is done by the insertion of a non-mutated gene DNA sequence into a DNA molecule. 

Making Diseases Cells Apparent to the Immune System 

Another type of gene therapy involves the body’s immune system . An example of this therapy is the use of checkpoint inhibitors. With this therapy, the immune system is modified to recognize material in the body that is produced by the mutated genes in order to destroy them and to prevent the illnesses they cause. 

Risks of Gene Therapy 

There are some known risks of gene therapy. So far, the most common problem associated with gene therapy is a lack of effectiveness. However, there are also adverse effects that may occur. 

Unwanted Immune System Reaction 

Gene therapy that involves the immune system may cause excess immune reactivity to healthy cells that resemble the disease cells, potentially causing damage to healthy cells.   

Wrong Target Cell

Potentially, the immune reaction that is mediated by gene therapy can affect the wrong cell type, instead of the intended target cells. 

Infection Caused by Viral Vector 

When a viral vector is used, there may be a risk that the virus could cause an infection. Depending on the primary disease that is being treated, a person receiving gene therapy may have a weak immune system and, therefore, could have difficulty fighting the virus. 

Possible Tumor

A new DNA sequence that’s inserted into a person’s genes could potentially lead to a mutation that could cause cancer to form. 

What to Expect From Gene Therapy 

If you are considering gene therapy, you will go through a process of diagnosis, treatment, and medical surveillance to assess the effects. 

This step will determine whether you have a medical condition that can be treated with gene therapy. This means that you would have a blood sample sent to a laboratory to identify treatable gene mutations that are associated with your medical condition.

Examples of conditions that may be treatable with gene therapy include:

• Cystic fibrosis : An inherited disorder in which thick mucus is produced, clogging the airways and blocking the secretion of digestive enzymes
• Sickle cell disease : An inherited disorder that results in abnormal hemoglobin production (the protein that carries oxygen in the red blood cells)
• Leber's hereditary optic neuropathy (LHON) : An inherited disorder that causes the death of cells in the optic nerve , resulting in damage to central vision
• Inherited or acquired retinal disease : Conditions that damage to the retina , the light-sensing layer in the back of the eye
• WW domain-containing oxidoreductase (WWOX) epileptic encephalopathy syndrome : A genetic condition resulting in severe epilepsy, developmental delays, and early death
• Spinocerebellar ataxia and autosomal recessive 12 (SCAR12) : An inherited disorder resulting in seizures in infancy, developmental delays, and inability to coordinate movement
• Cancer : Many types of cancer

Your treatment may involve collection of your cells and delivery of the genes into your cells with a viral vector or liposome. The modified cells will be restored to your body after the treatment. 

Surveillance

The effects of your treatment will be assessed, and you will be monitored for adverse events (side effects). If this occurs, you may be treated again. 

Clinical Trials

You can find clinical trials for gene therapy by talking to your doctor or by searching for organizations that support your medical condition, such as the Cystic Fibrosis Foundation.

Gene therapy is a relatively new treatment designed to alleviate disease by modifying defective genes or altering the production of proteins by faulty genes. There are several ways that healthy genes can be inserted into the body, such as inside a deactivated virus or inside a fat particle.

Sometimes immature and healthy cells are transplanted to replace cells that have a disease-causing mutation. This type of therapy can cause side effects, and there is also a risk that it might not work. 

A Word From Verywell 

If you have a genetic disease with a known and identified gene mutation, you might be a candidate for gene therapy treatment in a clinical trial. This type of treatment is not a standard therapy, and you would need to be monitored closely so that you and your doctors would know if the therapy is working and whether you are having any side effects.

You can talk to your doctor about gene therapy. This treatment is not widespread, so there is a possibility that you may need to travel to be able to participate in a clinical trial if there is not a research study near you. 

This therapy is considered safe, but there are risks and side effects. You may have an opportunity to participate in a clinical trial, and side effects and adverse effects would be monitored. 

One example of this therapy is the use of a deactivated virus to insert a portion of a DNA molecule into the body’s cells so that the healthy DNA sequence can provide a blueprint for healthy proteins. 

The main goal of gene therapy is to provide DNA or RNA to code for healthy proteins so the body will not be affected by a genetic disease. 

Cystic Fibrosis Foundation. Gene therapy for cystic fibrosis .

Food and Drug Administration. What is gene therapy?

Jiang J, Sun G, Miao Q, Li B, Wang D, Yuan J, Chen C. Observation of peripapillary choroidal vascularity in natural disease course and after gene therapy for Leber's hereditary optic neuropathy. Front Med (Lausanne) . 2021;8:770069. doi:10.3389/fmed.2021.770069

Repudi S, Kustanovich I, Abu-Swai S, Stern S, Aqeilan RI. Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes . EMBO Mol Med. 2021;13(12):e14599. doi:10.15252/emmm.202114599

Tan TE, Fenner BJ, Barathi VA, Tun SBB, Wey YS, Tsai ASH, Su X, Lee SY, Cheung CMG, Wong TY, Mehta JS, Teo KYC. Gene-based therapeutics for acquired retinal disease: Opportunities and progress . Front Genet. 2021;12:795010. doi:10.3389/fgene.2021.795010

Lu Z, Chen H, Jiao X, et. al. Germline HLA-B evolutionary divergence influences the efficacy of immune checkpoint blockade therapy in gastrointestinal cancer . Genome Med . 2021;13(1):175. doi:10.1186/s13073-021-00997-6

By Heidi Moawad, MD Dr. Moawad is a neurologist and expert in brain health. She regularly writes and edits health content for medical books and publications.

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Review article, the promise and the hope of gene therapy.

• 1 Department of Molecular Technologies and Stem Cell Therapy, Miltenyi Biotec, Bergisch Gladbach, Germany
• 2 Laboratory of Biology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

It has been over 30 years since visionary scientists came up with the term “Gene Therapy,” suggesting that for certain indications, mostly monogenic diseases, substitution of the missing or mutated gene with the normal allele via gene addition could provide long-lasting therapeutic effect to the affected patients and consequently improve their quality of life. This notion has recently become a reality for certain diseases such as hemoglobinopathies and immunodeficiencies and other monogenic diseases. However, the therapeutic wave of gene therapies was not only applied in this context but was more broadly employed to treat cancer with the advent of CAR-T cell therapies. This review will summarize the gradual advent of gene therapies from bench to bedside with a main focus on hemopoietic stem cell gene therapy and genome editing and will provide some useful insights into the future of genetic therapies and their gradual integration in the everyday clinical practice.

Introduction

The idea that a gene can be delivered into specific cell types and its expression can lead to therapeutic efficacy, dramatically improving the patients' quality of life, was originally introduced by Theodore Friedmann 45 years ago and was later strongly encouraged and realized by George Stamatoyannopoulos, one of the founding members of the American Society of Gene and Cell Therapy (ASGCT). In this setting, the drug, which in the case of gene therapy is a gene, is packaged within a vector used to facilitate its entrance into the patients' cells. Of course, the notion of gene therapy has evolved, and in general, we refer to gene therapy when a therapeutic process involves genetic manipulation of the patients' cells with the use of a nucleic acid. This is actually the most important difference between cell and gene therapy: in cell therapy, the cells are not genetically modified but instead are subjected to a certain manipulation involving cell culture and exposure to specific types of media whereas gene therapy is mediated by the addition of any nucleic acid. For obvious reasons, the idea of gene addition was particularly applicable in monogenic diseases based on the simplified notion of “adding the missing gene or the normal allele to compensate for the expression of the mutated allele.” However, under the view of the latest advancements, gene therapy does not correspond to an addition of a gene, otherwise missing in the patient's cells, but with a gene that could offer therapeutic benefit to the affected individual.

There are basically three types of gene therapy: ex vivo, in vivo , and in situ . In ex vivo gene therapy, the target cells are removed from the patient's body, engineered either by the addition of the therapeutic gene or by other genetic manipulations that allow correction of the phenotype of the disease. The “corrected” cells are subsequently re-infused to the patient. This type of intervention is also termed in vitro gene therapy and is particularly applicable to blood diseases: in the case of blood cancer, the target cell may be T and, most recently, NK cells, and the therapeutic gene is the chimeric antigen receptor (CAR). In the case of monogenic diseases, the target cell is the hemopoietic stem cell (HSC) and the transgene varies analogous to the disease. The viral vectors utilized in both cases are mostly retroviral vectors, belonging either in the lentiviral or the oncoretroviral families of Retroviridae . However, depending on the affected tissue, ex vivo gene therapy is not always the intended type of corrective approach. For example, if the target organ is the brain, the spinal canal, or the liver, another type of therapy is employed, termed in vivo gene therapy. In this setting, the therapeutic vector is administered systemically in the blood circulation or the cerebrospinal fluid of the patient, and depending on the disease, different types of viral vectors are utilized, such as adenoviral vectors (AVs) or adeno-associated viral vectors (AAVs). Finally, there is a last scheme of gene therapy, in which the viral vector is administered in situ , i.e., to a specific organ or area in the body of the patient either through direct injection, e.g., into the tumor (in the case of melanoma) or into suitable brain areas (in the case of neuropathies) or by an insertion of a catheter in the case that the organ to be treated is the heart. The selection of the procedure depends entirely on the type of indication, the affected tissue, and the cell type that requires correction. In contrast to HSCs, namely, CD34 + cells, that can be easily isolated from the patients, nerve stem cells are difficult to obtain for ex vivo manipulation. In addition, stem cells are only partially characterized in the liver. Hence, gene therapy for specific organs or indications is dependent on systemic or in situ administration of the therapeutic vector.

Although the idea of genome correction was quite innovative in its nature, especially during the 90s, clinical translation involving genome correction is still rare and adoption of the application of gene therapy at a wider scale and in the context of a medical routine has been only partial. To date, there are more than 2,600 clinical trials concerning gene therapy and/or genome editing, but very few therapeutic drugs have acquired marketing authorization for different indications (summarized in Table 1 ).

Table 1 . Gene therapy products that have acquired marketing authorization.

During the early times of its development, the gene therapy field has faced a lot of skepticism specifically after the unfortunate death of Jesse Gelsinger ( Teichler Zallen, 2000 ) but also later on during the leukemic events recorded on the X-SCID clinical trial ( Papanikolaou and Anagnou, 2010 ) in the early 2000s. The death of Jesse Gelsinger not only had a profound impact on the gene therapy field, it also underlined the general lack of knowledge about the vector–host interactions and ultimately pointed out the weak spots within the collaboration between the researchers and the regulatory agencies. Eventually, the case of Gelsinger has been quoted relatively recently for a number of times 1 ( Baker and Herzog, 2020 ) specifically in view of the coronavirus pandemic and the generation of a new and effective vaccine. Of note, one of these reports 1 correlates the safety issues raised around the time of Gelsinger's death with the genome editing approaches currently employed, rather successfully, by a number of companies and academic institutions.

The scientific community is characterized by a heterogeneity in terms of taking risks, since there are scientists who intensely question the safety of any novel therapeutic approach and scientists who pave the way toward innovative and frequently risky treatments. A striking example of such risks and their potential to shape the policies around genetic therapies has recently happened in China, where the regulatory norms originally comprised mostly technical management methods or ethical guidelines under a broad legal framework issued by Commissions of the State Council in combination with departmental regulations and regulatory documents issued by individual ministries ( Wang et al., 2020 ). It was only after the incidence with the CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeats) babies in November of 2018 ( Lander et al., 2019 ) that urged China to advance legislation in areas of biosecurity, genetic technology, and biomedicine. To this end, the “Biosafety Law” was approved in 2019 by the Standing Committee of the National People's Congress. The aim of this law is to become a basic, systematic, comprehensive, and dominant legal framework on biosafety. Therefore, the regulatory landscape in genetic therapies is currently being shaped in China.

On the other hand, in Europe and in the USA, any new drug, regardless if it is gene therapy related, is not judged by the number or even the quality of publications, but eventually by the regulatory authorities who have the legal capacity to determine the marketing authorization of the formulation. However, the regulatory authorities have different views from the researchers in terms of innovation and safety. It is also important to keep in mind that regulators are basing their decisions on data and always compare those to the pre-existing state of the art of a specific indication in terms of equivalency. Hence, any new therapy, from a regulatory aspect will be thoroughly investigated and examined on the safety profile it presents and eventually on the extent of comparability between the currently authorized therapeutic treatments. This approach is employed both by the European Medicines Agency (EMA) in Europe and the Food and Drug Administration (FDA) in the United States ( Iglesias-Lopez et al., 2020 ).

One strategy that can be utilized by regulators, including governments, health technology assessment (HTA) bodies, and health care decision makers, in order to advance and promote the development of novel medicinal treatments, is to recognize and award innovation. In the review of De Solà-Morales et al. (2018) , the authors try to investigate how innovation is defined with respect to new medicines. Their conclusion is that innovation is differentially defined through countries, depending on independent political and societal factors. Hence, it is challenging to achieve common alignment, although coordination between countries and among regulators should be strongly encouraged as it would eventually help researchers and/or manufacturers toward determining mutually applicable research policies that can drive innovation. In their review ( De Solà-Morales et al., 2018 ), components and dimensions of innovation are mentioned and include notions such as unmet need, health outcomes, novelty, step change, availability of existing treatments, efficacy, new molecular entity, molecular novelty, therapeutic value, market share, cost-saving, disease severity, clinical benefit, safety, pharmacological/technological differences from current treatments, etc.

Innovation in other industrial sectors is defined usually as any improvement of the end product either in terms of manufacturing or in terms of cost reduction in the long term. However, this usually does not apply in the health care sector: a new product is often substantially different from existing therapies and improvements in patients' quality of life; i.e., the therapeutic benefit, as a result of the application of the innovative approach, is of the greatest importance. Another major aspect is the overall expenditure associated with development of the novel approach by the health industry, which is usually high and is currently the focus of specific discussions in Europe and in the United States and ultimately points toward the affordability within the public health budgets ( McCabe et al., 2009 ). However, as considerations about the costs are not usually included during the original design of the novel approach as a key component of innovation, there is the probability that innovation in pharmaceuticals and cell/gene therapy may not be aligned with the requirements of public or insurance health budgets and by extrapolation of society as a whole. Specifically for gene therapy approaches, the term “financial toxicity” is already circulating among the policy makers, the industry, and, consequently, the researchers.

Paradigms of definitions of innovations in different European countries are listed below: In France, the HAS (Haute Autorité de Santé) defines innovative products as those for which the producers assert a medium to major improvement of the clinical benefit compared to the currently available treatments [i.e., Amélioration du Service Médical Rendu (ASMR) of level I, II, or III] ( O'Connor et al., 2016 ). Other agencies such as the Swedish Tandvårds–och läkemedelsförmånsverket (TLV), the Scottish Medicines Consortium (SMC), and the National Health Service in England (NHS) take into consideration the novelty of the approach but in combination with the improvement of patients' quality of life and any potential reformation of the health care system; i.e., the new therapeutic approach should present palpable added value ( De Solà-Morales et al., 2018 ). Surprisingly, in the Netherlands, the Zorginstituut Nederland (ZINL) characterizes a product as innovative when it seems to be promising from a scientific point of view, but for which even insufficient data can overall provide a reasonably positive outlook and consequently effect a constructive response by the agency ( De Solà-Morales et al., 2018 ). Finally in Germany, innovation is not referenced within the legal framework and in general the focus lies on the additional therapeutic benefits provided by the novel approach ( De Solà-Morales et al., 2018 ).

To summarize, the definition of a novel medical approach as innovative in essence lies in its truly innovative nature. However, ideally, it should combine additional features such as (a) be at least as safe as the current treatments, (b) dramatically improve the patients' quality of life, and (c) be affordable by reimbursement bodies (payers). Finally, another important aspect would be to distinguish between price and the true value of the novel approach.

Early Insights From Commercialization of Gene Therapies in Europe

Toward a better understanding of the impact that gene therapy presents at a societal level, one should keep in mind that in terms of innovation, any gene therapy approach is considered highly innovative. Consequently, in the context of genome editing, identification of the nucleases that generate targeted double strand DNA breaks that can, in a subsequent process, be repaired by indels or via homologous recombination and correct any genetic mutation was not only innovative but also considered a scientific breakthrough.

However, any marketing authorization of these products is expected to be scrutinized by the regulatory agencies as it was previously the case for other gene therapy products. Under the existing regulatory framework, cellular products that have been subjected to more-than-minimal manipulation are broadly classified as either medicinal products (EU) or biologics (USA). In Europe, cell-based medicinal products are regulated under the Advanced Therapy Medicinal Product (ATMP) Regulation, which mandates that all ATMPs are subject to a centralized marketing authorization procedure ( Coopman, 2008 ). All marketing authorization applications are subject to a 210-day assessment procedure by the EMA, supported by the Committee for Advanced Therapies (CAT), before a license can be granted. Member states retain responsibility for authorization of clinical trials occurring within their borders and have the option to exempt certain products used on a non-routine basis for unmet clinical need, referred to as the “Hospital Exemption” based on Article 28 of Regulation (EC) 1394/2007. As with all medicines, the EMA continues to monitor the safety and efficacy of ATMPs after they are approved and marketed and provides scientific support to developers for designing pharmacovigilance and risk management strategies used to monitor the safety of these medicines.

Regulatory approval, however, does not guarantee availability to patients or reimbursement by European health systems, because novel therapies, regardless of their mechanism of action, have to undergo formal Health Technology Assessment ( Touchot and Flume, 2017 ). From a time perspective, the first marketing authorization for gene therapy products for rare diseases occurred in 2012 with Glybera ® (EU), followed by Imlygic ® (EU and USA) in 2015 and Strimvelis ® (EU) in 2016. Therefore, these products have not only undergone meticulous evaluation from regulatory agencies, they have been also subjected to Health Technology Assessment by the reimbursement bodies and have received positive opinions from regulators and payers, and thus, a comprehensive analysis of their life cycle can now be conducted.

Glybera (alipogene tiparvovec) was the very first gene therapy agent to officially receive marketing authorization in Europe for treatment of lipoprotein lipase deficiency, a deadly disease causing severe pancreatitis to the affected patients. LPL deficiency (LPLD) is classified as a rare disease, estimated to occur in ~1 in 250,000 people in the general population and has been described in all races. Glybera was an adeno-associated serotype 1 vector (AAV-1), designed to deliver in vivo to the patients several copies of the normal allele (gene addition) by injection to several parts of the muscle areas of the body. Each vial of the vector had an estimated cost of ~100,000 euros, and to achieve a therapeutic quantity in the body of the patient, it was necessary to inject at least 10 vials. This fact raised the price of the therapy to 1 million euros. The drug was originally marketed by uniQure and, after going through formal evaluation through Health Technology Assessment in Germany and in France, failed to achieve a recognition of benefit in either country ( Touchot and Flume, 2017 ). In France, the HAS Transparency Commission stated that ( Touchot and Flume, 2017 ):

➢ “A moderate effect on triglycerides and on episodes of pancreatitis has been observed but this effect was not sustained in the medium–and long-term” (in line with submitted efficacy data showing only transient efficacy);

➢ “The clinical relevance of the chosen primary efficacy endpoint (reduction in the triglyceride level) is debatable;”

➢ “Uncertainties about the short–and medium-term safety of this gene therapy, which cannot be re-administered because of its action mechanism, remain.”

As a result, the HAS concluded that the actual benefit of Glybera is insufficient to justify reimbursement by the French national health insurance and thus the product was not commercialized in France.

In Germany, it was initially assessed as a community product but was evaluated by AMNOG (the German Health Technology Assessment process) to confer “unquantifiable additional benefit” because of lack of proper clinical data that would adequately justify the actual therapeutic potency of the product ( Touchot and Flume, 2017 ). This led to a repositioning of the drug to a hospital-only product and allowed price negotiations directly between hospitals and payers. In the case of Germany, these discussions were fruitful only for a single patient that was treated at Charité in Berlin in September 2015 with an estimated price of 900,000 euros after an agreement with DAK (Deutschen Angestellten-Krankenkasse), a large German health insurance provider. This patient, was a woman with LPLD who suffered consecutive debilitating pancreatitis and was hospitalized in intensive care more than 40 times, and thus, she qualified for gene therapy because of the severity of her overall clinical status. The woman was fully cured and never suffered from pancreatitis again ( Crowe, 2018 ). Despite these hopeful events and taking into account the very low number of patients, uniQure decided in 2015 not to apply for approval in the USA and exclusively licensed rights in Europe to Chiesi Farmaceuticals for €31 million ( Regalado, 2016 ). A total of three remaining doses left on the shelf were basically given away in one patient from Italy and two German patients who received doses for 1 euro each. Since October 2017, the utilization of Glybera was discontinued in EU because marketing authorization to Chiesi Farmaceuticals was not renewed, for financial reasons.

Imlygic (Talimogene laherparepvec), which has been authorized for treatment of melanoma, is a vector based on a strain of Herpes Simplex Virus 1 (HSV-1) that possesses oncolytic properties in combination with the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) to attract antigen-presenting cells (APCs) in the affected area. Upon administration in situ , Imlygic lyses tumor cells, enhances antigen loading of MHC class I molecules, and express GM-CSF to increase tumor antigen presentation by dendritic cells ( Conry et al., 2018 ). Therefore, although it is administered in situ , it provokes a systemic anti-tumor immunity. It was approved by the EMA and FDA in October and December of 2018, respectively ( Touchot and Flume, 2017 ). Upon approval of the regulatory agencies, evaluation of Imlygic has been completed so far in the UK ( Touchot and Flume, 2017 ). Initially, the NICE (National Institute of Clinical Excellence) concluded that Imlygic, despite its truly innovative mechanism of action, was not cost-effective and did not confer significant advantage in terms of the overall survival of the patients compared to the existing therapies for melanoma. This evaluation prompted the company to discuss a respective discount with the Department of Health ( Touchot and Flume, 2017 ), to agree to a patient access scheme, and to narrow the indication of coverage to patients who did not qualify for systemically administered immunotherapies. Imlygic is currently still being evaluated in Germany by IQWiG (the German health technology assessment body) and the Federal Joint Committee (G-BA), which requested additional data to complete the assessment including comparison with administration of GM-CSF alone. Of note, previously in clinical trials, the overall response rate (ORR) was increased in the Imlygic arm (26.4%) compared to the GM-CSF arm (5.7%). The mean overall survival (OS) was 23.3 months in the Imlygic arm, vs. 18.9 months on the GM-CSF arm ( p = 0.051), showing a marginal statistical trend in favor of Imlygic ( Andtbacka et al., 2015 ). However, administration of GM-CSF is also not an authorized treatment for melanoma. This poses a risk toward the final positive evaluation of Imlygic as it could be again classified as providing “no quantifiable additional benefit,” suggesting that it is probable that it will face challenges in reaching a wider number of patients, unless newly generated data provide an undisputable therapeutic benefit compared to the standard treatment, as this is defined by each individual payer ( Touchot and Flume, 2017 ).

The aforementioned products are employed in in vivo and in situ gene therapy, respectively. However, one of the greatest achievements in the history of the field was the case of Strimvelis ® . Strimvelis ® , is a product derived from genetic engineering of HSCs isolated from patients suffering from severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID). In this case, genetic correction of HSCs is mediated by gene addition of the normal allele packaged inside an oncoretroviral (also termed gamma retroviral) vector. In terms of safety, the gene therapy field has been severely hampered by the unfortunate leukemic events that occurred during the clinical trial for another form of SCID, namely, the X-SCID. In the early 2000s, four cases of leukemia in the French X-SCID clinical trial were recorded out of the initial seven infants that were recruited for the study. These events were attributed to the vector's integration into the proto-oncogene LMO2 ( Hacein-Bey-Abina et al., 2003a ; Kohn et al., 2003 ) and triggered a new field of research resulting in a comprehensive characterization of the preference to integrate of lentiviral vectors and oncoretroviral vectors ( Montini et al., 2006 ; Biasco et al., 2017 ) within the human genome. Surprisingly, although lymphoproliferative aberrations were also observed in the trials of HSC gene therapy for Wiskott–Aldrich syndrome ( Braun et al., 2014 ) and for chronic granulomatous disease (CGD, Stein et al., 2010 ), no case of leukemic events for ADA-SCID in the context of clinical trials has been recorded, despite the fact that all the aforementioned indications employed oncoretroviral vectors. Unfortunately, 4 years after Strimvelis ® received marketing authorization, lymphoid T cell leukemia has been reported in one patient in October of 2020, and its relationship to the gene therapy is currently under investigation ( Ferrari G. et al., 2020 ). Strimvelis ® was originally developed in Ospedale San Raffaele in Milan ( Aiuti et al., 2002 , 2009 ) in collaboration with Fondazione Telethon before it was acquired by GlaxoSmithKline and, in May 2016, received approval in Europe. GSK initially collaborated with MolMed a clinical biotech company, to develop a robust process for commercializing the product. Because Strimvelis ® contains essentially HSCs that need to be engineered within a very short period of time (not more than 2 days), until today, it was authorized only in Italy (MolMed) and patients from other European countries are supposed to travel to Italy to receive the treatment ( Touchot and Flume, 2017 ). The Italian medicines agency (AIFA) agreed to a reimbursement of 594,000 euros based on the substantial clinical benefit for the patients in combination with the overall amount spared from a lifetime treatment with enzyme replacement therapy, as Strimvelis was beneficial for the public health budget in the long run ( Touchot and Flume, 2017 ). In 2018, GSK transferred all the assets associated with Strimvelis to Orchard Therapeutics ( Paton, 2018 ). Although the product has to undergo evaluation also in other European countries, and despite the small number of patients treated so far, it should be mentioned that the short time period between the approval and the reimbursement decision by the Italian authorities indicates that good clinical practice, good manufacturing practice, and robust clinical data combined with reasonable pricing can pave the way toward integrating gene therapies in medical routine. Of course, the report of the leukemic event is expected to create delays toward authorization in other countries until the results of the investigations are announced.

Last but not least, another important achievement for HSC gene therapy is Zynteglo ® , which received marketing authorization for treatment of transfusion dependent β-thalassemia (TDT), a disease that was the first candidate for HSC gene therapy. Significant research efforts toward the generation of erythroid-specific globin expressing lentiviral vectors were employed that were eventually successfully translated to clinical trials in 2006 ( Ferrari G. et al., 2020 ). Zynteglo ® , similar to Strimvelis ® , is a product derived from genetic engineering of HSCs isolated from patients suffering from TDT, transduced with BB305 lentiviral vector, which encodes a β-globin transgene (βT87Q globin), which also has antisickling properties. The results of phase I and phase II trials were reported and showed that gene therapy was efficacious in 80% of patients with non-β 0 /β 0 genotypes and 38% of patients with β 0 /β 0 genotypes, measured by transfusion independence at the 2-year follow-up ( Ferrari G. et al., 2020 ), while the rest of the participants reached various levels of transfusion reduction. On the basis of these results, Zynteglo ® received conditional marketing authorization for use in patients with transfusion-dependent β-thalassemia with non-β 0 /β 0 genotypes in 2019 in Europe, while the respective authorization by the FDA is still pending.

Undisputable success stories in the field of CAR-T gene therapy are also Kymriah and Yescarta. However, Zynteglo ® , Kymriah ® , and Yescarta ® have relatively recently received regulatory approval, and their assessment in terms of reimbursement is currently ongoing in EU and USA.

Excellent Science and Safety

Gene therapy based on viral vectors utilizes the natural ability of viruses to deliver genetic material to cells, and a large part of research has been devoted toward generating novel, more efficient, and safer delivery tools employing gammaretroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Retroviruses are particularly applicable in the case of HSC gene therapy because they have the unique capability to fully integrate their genome intact into the genome of the host cell. However, as with any new therapeutic approach, gene transfer using viral vectors also introduced new side effects. One of these side effects, known as insertional mutagenesis or genotoxicity, involves activation of proto-oncogenes or disruption of tumor suppressor genes due to retroviral vector integration. Of course, genotoxicity is a natural phenomenon that has been described since the discovery of retrotransposons, as transpositions of Long Interspersed Nuclear Elements (LINEs) were (i) detected as de novo insertions into the coding regions of factor VIII gene resulting in hemophilia A, (ii) integrated into the adenomatous polyposis coli tumor suppressor gene causing its disruption and generating colon cancer, (iii) detected into the myc locus in a breast cancer, and (iv) inserted into exon 48 of the dystrophin gene ( Löwer et al., 1996 ). These transpositions were detected in extremely very low frequency within the overall population and even within the population suffering from these specific indications. Regarding the utilization of retroviral vectors into gene therapy protocols, although the possibility of insertional mutagenesis was originally discussed as theoretically possible, such risks had been estimated to be extremely low, based on (a) the fact that over 90% of human genome is non-coding and (b) the assumption that proviral integration into the human genome would be random ( Papanikolaou et al., 2015 ). Unfortunately, these hypotheses were not verified after the reports of lymphoproliferation due to insertional activation of the LMO2 gene following gene therapy in the French X-SCID clinical trial ( Hacein-Bey-Abina et al., 2003a , b ), the leukemias developed in the Wiskott–Aldrich gene therapy trial ( Braun et al., 2014 ), and myelodysplasia attributed to EVI1 activation after gene therapy for CGD ( Stein et al., 2010 ). All these events highlighted the importance of understanding the underlying mechanisms that are responsible for integration into the preferred genomic loci but also the components that contribute toward the repair of the genome during the integration events. From a phenotypic standpoint, this lack of knowledge was translated as leukemic events only during clinical trials, as such events were not detectable during the pre-clinical development of gene therapies of the aforementioned indications. From a regulatory standpoint, the clonal dominance observed during the French β-thalassemia trial ( Cavazzana-Calvo et al., 2010 ) led to a clinical hold of the specific trial as per FDA guidelines for 5 years, until it was clear that the respective clonal dominance did not evolve to any kind of dysplasia or leukemia and it was safe to proceed and recruit more patients to the study.

All the aforementioned cases underlined the non-random integration patterns of retroviral vectors and sparked the field's interest toward characterizing the potential mechanisms. Therefore, it was comprehensively shown that gammaretroviral vectors preferentially locate around transcription start sites while HIV-based vectors strongly favor integration in transcriptional units and gene-dense regions of the human genome ( Papanikolaou et al., 2015 ). These properties rendered lentiviral vectors safer for gene therapy approaches compared to oncoretroviral vectors and paved the way toward substitution of oncoretroviral by lentiviral vectors. Indeed, lentiviral vector-based gene transfer into HSCs has subsequently been applied in the treatment of X-linked adrenoleukodystrophy ( Cartier et al., 2009 ), metachromatic leukodystrophy ( Biffi et al., 2013 ; Sessa et al., 2016 ), and Wiskott–Aldrich syndrome ( Aiuti et al., 2013 ) without any vector-related adverse events. Therefore, the clonal dominance observed in the β-thalassemia trial is still an open question regarding whether this was purely coincidental or was truly attributable to a clonal proliferation as a result of the HMGA-2 dysregulation.

Aside from the comprehensive characterization of the integration preference of onco- and lentiviral vectors, the field furthermore strengthened the efforts toward making gene therapy safer by generation of self-inactivating (SIN) vectors. Because activation of the LMO2 oncogene was attributed to the strong enhancer elements within the U3 region of the retroviral Long Terminal Repeats (LTRs) ( Hacein-Bey-Abina et al., 2003a ), part of the U3 enhancer was removed in order to minimize the probability of activating neighboring oncogenes. In addition, alternative genetic elements, such as chromatin insulators, were gradually incorporated in the remaining U3 region of the LTR. Chromatin insulators are DNA sequences capable of maintaining the expression of a gene region independently of the expression of the neighboring gene region, by inhibiting their natural interactions (insulation). Insulator sequences have two main characteristics: (a) barrier activity, i.e., gene expression of a chromatin region is not affected by the adjacent heterochromatin region if an insulator is inserted between them, and (b) enhancer blocking activity, i.e., inhibition of the concerted action between a promoter and an adjacent enhancer ( Heger and Wiehe, 2014 ). Therefore, the incorporation of a chromatin insulator into the U3 region of the LTR, on the one hand, offers additional protection against the activation of neighboring oncogenes and, on the other hand, ensures the expression of the therapeutic gene in case of integration in a heterochromatic region. For globin gene therapy, significant efforts have been employed to this end due to long-standing knowledge that the expression of globin genes was variable due to the integration of the vector into transcriptionally inactive regions of chromatin, i.e., dependent on “position effects” ( Persons et al., 2003 ). Additional efforts deriving from the group of Dr. Stamatoyannopoulos have demonstrated the need to incorporate chromatin insulators into vectors intended for the gene therapy of hemoglobinopathies ( Aker et al., 2007 ). However, later studies showed that incorporation of chromatin insulators leads to a significant loss of titer of the lentiviral vector ( Urbinati et al., 2009 ), which typically translates to greater manufacturing costs as more vector is necessary to achieve the ideal transduction efficiency that would suffice to exhibit therapeutic efficacy. Currently, in the ongoing clinical trials of bluebird bio, the globin vector utilized is insulator-free ( Negre et al., 2015 ), and as previously stated, it remains unclear whether the initial clonal dominance was because of the higher proliferation rate of a specific clone as a result of the vector integration into the HMGA gene or whether this observation merely reflects the effects of incorporating a limited number of genetically modified hematopoietic stem cells into the patient's marrow. Thus, bluebird's vector format is not considered dangerous from a regulatory standpoint.

Excluding genotoxicity, in clinical protocols that utilize lentiviral vectors, the regulatory agencies are also concerned about recombination events that might occur during the manufacturing process of the vectors and require extensive data demonstrating the lack of replication-competent retroviruses or lentiviruses (RCRs/RCLs) partly because the agencies assume higher probability for genotoxicity if RCRs or RCLs are present ( Milone and O'Doherty, 2018 ). In addition, they request long-term follow-up monitoring of the patients participating in cell and gene therapy studies for the presence or RCRs/RCLs, new incidence or re-appearance of autoimmune, rheumatologic, and neurological disorders, or delayed malignancies, as a result of genotoxicity. Toward generating safer tools to reduce the risk of insertional mutagenesis, integration-deficient lentiviral vectors (IDLVs) or non-integrating lentiviral vectors (NILVs) have been generated ( Wanisch and Yáñez-Muñoz, 2009 ; Milone and O'Doherty, 2018 ), which present lower probability of causing either genotoxicity or generating RCRs. Unfortunately, their use is rather limited because they provide merely transient transgene expression in proliferating cells, but they can still be employed to promote stable expression in non-dividing cells or to induce RNA interference and mediate homologous recombination ( Wanisch and Yáñez-Muñoz, 2009 ).

To summarize, clinical trials in gene therapy via gene addition were initiated in the early 1990s, and until the late 2010s, a significant amount of effort combining excellent science and extensive assessment of potential risk factors have managed to make gene therapy more robust and simultaneously achieve great advancements toward clinical benefit.

The Era of Genome Editing: Challenges and Prospects

Over the last decade, the discovery of important novel regulatory elements of the human genome, combined with the continuous developments of novel technologies in the field of molecular biology and biotechnology, has conferred important conceptual insights for the implementation of new molecular approaches for the treatment of monogenic disorders. The advent of induced pluripotent stem cells and the design of novel nucleases that target specific areas in the genome have rendered gene editing approaches pivotal players in the field of therapy of inherited diseases. Gene targeting that is currently mediated by genome editing, is anticipated to outperform the classical approach of gene therapy via gene addition utilizing retroviral vectors, mainly due to the inability of the latter to establish targeted vector integration into the host genome.

Gene editing technology allows site-specific genome modifications, ranging from single-nucleotide edits to large deletions/inversions or targeted integration of entire genes, and is anticipated to outperform the classical approach of gene therapy via gene addition utilizing retroviral vectors, in part due to the inherent risk of insertional mutagenesis of gene addition by retroviral vectors and its limitations to treat gain-of-function mutations or defects in large genes. Moreover, in contrast to gene addition, most gene editing approaches maintain the natural genomic regulation of the gene of interest and thus physiological expression.

The original and still most prevalent application of gene editing for therapy relies on double strand breaks in DNA, which are introduced by engineered nucleases that act at predetermined and targeted genomic loci ( Genovese et al., 2014 ). Such nucleases are:

• Zinc Finger Nucleases (ZFNs)

• Transcription Activator-Like Effector Nucleases (TALENs)

• Cas nuclease of the CRISPR/Cas9 system.

Their mode of action is to induce a double strand break (DSB) on the DNA molecule followed by respective repair either through the non-homologous end joining (NHEJ) or via homologous recombination (HR). Through NHEJ, repair of the DSBs leads to disruption of the target sequence by generation of small insertions or deletions, which collectively are called “indels.” Repair through HR leads to full reconstitution of the target sequence if a template donating a homologous sequence, that serves as a matrix for the repair to take place, is provided.

It should be noted, however, that a DSB is actually the initiating step in natural genome editing and occurs in mammalian cells on several occasions, such as the V(D)J recombination through the RAG1/RAG2 enzymes ( Jasin and Rothstein, 2013 ), during the meiotic recombination mediated by the Spo11 nuclease ( Jasin and Rothstein, 2013 ) and finally during the natural gene drives, managed by homing endonucleases ( Burt, 2003 ). Also, all mammalian cells possess robust DNA repair mechanisms; however, the frequency of repair either through NHEJ or HR increases at least by a 100-fold following a double strand break ( Jasin and Rothstein, 2013 ). Therefore, the novel engineered nucleases are necessary to achieve adequate gene correction to reach the anticipated therapeutic levels required. This aspect is of particular interest for the clinical applications of engineered HSCs, because the number of CD34 + cells that need to be infused to patients are in the range of 5 × 10 6 -10 7 cells/kg and 80% of those should be genetically corrected. For example, for a thalassemic patient with an average weight of 70 kg, one would need to infuse 5 × 10 6 × 70, i.e., a total of 3.5 × 10 8 viable CD34 + cells, of which at least 80% should be genetically corrected. Thus, in order to have a final total cell count of ~4–5 × 10 8 cells in the final cell product, it is anticipated that optimization toward mobilization of HSCs to the periphery specifically from patients suffering from rare diseases, optimization of infusion protocols, as well as optimization of the editing process per se are absolutely necessary. These are current challenges that will increasingly appear as we pave the way toward clinical genome editing applications. For example, even optimized transfer of nucleases by electroporation leads to a significant loss of cell viability, which, in turn, necessitates efficient mobilization and collection of high numbers of HSCs as editing substrate. Unfortunately, because in certain cases, such as in the case of sickle cell disease, patients mobilize poorly or due to innate characteristics of the disease per se use of granulocyte colony stimulating factor (G-CSF) is not recommended, one of the first challenges toward clinical translation would be the existence of a validated freezing protocol followed by a validated thawing protocol as it is possible that certain patients would need to undergo multiple rounds of mobilization.

A second notable challenge is the process of genome editing in terms of culture conditions including media, cytokines, timelines, and inclusion of several means of molecules or strategies to enhance the efficiency of the editing. To this end, several amendments have been published. Dever et al. (2016) reported a CRISPR/Cas9-based gene editing approach that combines Cas9 ribonucleoproteins (RNPs) and delivery of a homologous template via an AAV to achieve homologous recombination at the β-globin gene in HSCs combined with a concomitant purification method that generates a population of hemopoietic stem and progenitor cells with more than 90% targeted integration. Respective results were also obtained for SCID-X1 ( Pavel-Dinu et al., 2019 ) following the same approach, i.e., the CRISPR/Cas9-AAV6-based strategy to insert the cDNA of the normal gene into the endogenous start codon. This approach aims to functionally correct disease-causing mutations throughout the genomic locus. Unfortunately, a similar strategy could not be employed for hemoglobinopathies as the presence of genomic introns is mandatory to achieve tissue specificity as well as therapeutic expression levels ( Uchida et al., 2019 ).

Another interesting approach to achieve higher editing efficiency is to modulate the cellular pathways responsible for DSB repair. More specifically, the efficiency of HR by genome editing is limited by DSB repair pathways that compete with homology-directed repair (HDR), such as non-homologous end joining (NHEJ) ( Nambiar et al., 2019 ). The choice of the type of the DSB repair pathway is mostly determined by the DSB resection, a nucleolytic process that converts DSB ends into 3′-single-stranded DNA overhangs ( Nambiar et al., 2019 ). Certain NHEJ factors, including 53BP1, promote the direct joining of DSBs by protecting DNA ends from resection. Limited resection of DSB ends can expose regions of sequence microhomology, which favor DSB repair through microhomology-mediated end joining (MMEJ), while more extensive DSB resection generates the long 3′-single-stranded DNA tails required for HDR ( Nambiar et al., 2019 ). Thus, cellular factors that impede DSB resection represent major barriers to HDR-mediated precision genome editing. Toward this direction, the authors characterized RAD18 as a stimulator of CRISPR-mediated HDR and identified its mechanism of action that involved suppression of the localization of the NHEJ-promoting factor 53BP1 to DSBs ( Nambiar et al., 2019 ).

An alternative strategy to enhance the efficiency of genome editing was to transiently silence p53 ( Schiroli et al., 2019 ). More specifically, Schiroli et al. challenged the successful use of the combination of AAV and generation of DSBs by engineered nucleases such as ZFNs and CRISPR/Cas9 by claiming that they cause excessive DNA damage response (DDR) across all hemopoietic stem and progenitor cell subtypes analyzed ( Schiroli et al., 2019 ). DDR consequently induced cumulative p53 pathway activation, constraining proliferation, yield, and engraftment of edited HSPCs, which could be overcome by transient inactivation of p53. Of note, DDR is reported to be activated also under conditions of viral infections or vector transduction as there are recent reports correlating immune responses within the cells that undergo DNA damage ( Piras et al., 2017 ; Dunphy et al., 2018 ). Immune responses have also been detected in the context of gene therapy via gene addition ( Papanikolaou et al., 2015 ) after transduction of CD34 + cells with a GFP encoding lentiviral vector. It is not unprecedent that such immune responses are linked to DNA damage repair mechanisms, since retroviral integration presupposes breaks on the DNA chain. However, it should be noted that DDR is not always activated: For example, in the study by Papanikolaou et al. (2015) , transduction with the GFP lentiviral vector activated immune responses without significant DDR. On the contrary, in the study by Piras et al. (2017) , there was significant upregulation of DDR. One important difference between the two studies was the multiplicity of infection (MOI); in the first study, an MOI = 10 was employed, while in the second study, the authors experimented with MOI = 100. These results immediately suggest that the MOI plays a crucial role during the manufacturing process since both studies employed a VSV-G pseudotyped GFP encoding lentiviral vector and used cord blood CD34 + cells. Obviously, a better understanding of the interplay between vectors or nucleic acid molecules with the host cell in terms of both quality and quantity would be necessary to advance the field of gene engineering. An important aspect that is linked to clinical translation is that activation of vector-mediated DDR can induce significant, albeit mild, increase in apoptosis of human HSCs in culture ( Piras et al., 2017 ), which typically results in lower engraftment of engineered HSCs in vivo , particularly during the early phases of hemopoietic reconstitution ( Piras et al., 2017 ; Piras and Kajaste-Rudnitski, 2020 ). Therefore, induction of DDR mechanisms in the context of genome editing should be taken into serious consideration, and strategies toward achieving robust and efficient editing without interfering with the stem cell-like character of CD34 + should be generated.

As reviewed by Piras and Kajaste-Rudnitski (2020) , HSCs have devised several strategies of responding to RNA molecules as well as ssDNA and dsDNA molecules. Indeed, a plausible approach to increase the efficiency of retroviral transduction or gene editing would be to assess the mechanisms of innate immunity and nucleic acid sensing in HSCs and harness their potential. For example, transient silencing of cellular nucleic acid sensors could increase the level of transduction or the efficiency of the editing. To that end, many researchers have focused on several transduction enhancers such as 16,16-dimethyl prostaglandin E2 (PGE2) and LentiBOOST™, poloxamers, the polycationic protamine sulfate, cyclosporine A and cyclosporine H, and rapamycin ( Piras and Kajaste-Rudnitski, 2020 ). PGE2 and LentiBOOST™ are already employed in the context of clinical trials ( Tisdale John et al., 2018 ), but it should be emphasized that the exact mechanism of action of the majority of these transduction enhancers is not fully elucidated. Besides the employment of transduction enhancers, additional strategies exist in terms of culture conditions that urge HSCs to move toward the S phase of the cell cycle in order to increase the successful HR. One strategy employed by Ferrari S. et al. (2020) was to transiently downregulate p53 with GSE56 in addition to including the E4orf6/7 protein of adenovirus, a known interactor with cellular components involved in survival and cell cycle ( Ferrari S. et al., 2020 ) to successfully enhance the efficiency of editing. From another perspective toward advancing safety, Wiebking et al. (2020) disrupted the uridine monophosphate synthetase (UMPS) involved in the pyrimidine de novo synthesis pathway rendering proliferation dependent on external uridine and providing thus the possibility to control cell growth by modulating the uridine supply. However, it should be noted that disruption of UMPS would be an additional genome editing process on top of any other correction, suggesting that to manufacture cell products that have been genetically engineered and present advanced safety features, one would have to edit at least two genomic loci. Although both strategies ( Ferrari S. et al., 2020 ; Wiebking et al., 2020 ) certainly assume great potential, they involve genetic manipulation beyond the current state of the art, and the transition to the clinic will probably be challenging from a regulatory standpoint.

A final aspect of great importance is the type of mutations that are introduced in the human genome in the context of therapy. One idea would be to add the desired transgene into a safe harbor. Papapetrou et al. (2011) characterized as safe harbors specific genomic loci based on their position relative to contiguous coding genes, microRNAs, and ultraconserved regions. Genomic safe harbors should fulfill the following criteria: (i) distance of at least 50 kb from the 5′ end of any mapped gene, (ii) distance of at least 300 kb from any cancer-related gene, (iii) distance of at least 300 kb from any microRNA (miRNA), (iv) location outside a transcription unit, and (v) location outside ultraconserved regions (UCRs) of the human genome (i.e., enhancers, exons, regulatory sequences, etc.). The idea is promising and has been widely employed in the context of induced pluripotent stem cells, and most lately, it was capitalized by Gomez-Ospina et al. (2019) toward showing therapeutic benefit for Mucopolysaccharidosis type I by generating a CRISPR/Cas9 approach that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34 + hematopoietic stem and progenitor cells. The authors demonstrated adequate therapeutic efficacy in an immunocompromised mouse model of Mucopolysaccharidosis type I and showed that the modified cells could secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and provide biochemical and phenotypic improvement in vivo .

Therefore, one approach is to introduce the therapeutic transgene into a safe harbor locus. Another approach is to introduce the therapeutic gene exactly in its natural position in the genome, thus ensuring lifelong regulation by the naturally occurring expression modulating elements affecting the respective region. This was already described to treat X-SCID 1 ( Pavel-Dinu et al., 2019 ) but is a particularly plausible approach for hemoglobinopathies aiming to correct either mutations within the β-globin gene in the case of β-thalassemia, or the specific point mutation for sickle cell disease. To that end, at least two successful strategies have been developed aiming to correct the IVS I-110 (G>A) mutation in β-thalassemia ( Patsali et al., 2019 ) via either CRISPR/Cas9 or TALENS or the sickle cell mutation ( Park et al., 2019 ). However, the most widely employed approach applicable for both sickle cell disease and thalassemia is the induction of fetal hemoglobin via genome editing. In 2013, the group of Stewart Orkin mapped a regulator of expression of BCL11A specific for the erythroid lineage ( Bauer et al., 2013 ), and a follow-up study employing genome editing proved that targeted disruption of the critical GATA1 binding motif within the +58 intronic BCL11A enhancer leads to indel generation and thereby to reduced BCL11A expression with associated induction of γ-globin expression in erythroid cells ( Wu et al., 2019 ). This notion was moved to the clinic by two ongoing clinical trials, NCT03745287 by CRISPR Therapeutics and NCT03653247 by Bioverativ. The two trials differ in the designer nucleases used to target the enhancer in that CRISPR Therapeutics utilizes a CRISPR approach, while Bioverative utilizes a ZFN. Regarding the CRISPR trial, short-term results of 15–18 months of follow-up reported two patients, one with thalassemia and a second with sickle cell disease, who demonstrated significant increase in hemoglobin values (expressed in g/dl) after gene therapy, combined with the presence of over 95% F-cells in peripheral blood ( Frangoul et al., 2020 ). This recapitulation of the HPFH (Hereditary Persistence of Fetal Hemoglobin) phenotype has become a common approach and was also employed as a therapeutic alternative by other researchers as well, first by disrupting the BCL11A binding motifs in the promoters of γ-globin genes by CRISPR ( Métais et al., 2019 ) or TALENs ( Lux et al., 2018 ), so as to inhibit the binding of BCL11A and hence prevent the silencing of γ-globin and also by comparing disruption of different HbF repressors, including KLF1 ( Lamsfus-Calle et al., 2020 ) and LRF ( Weber et al., 2020 ). Finally, efforts to reconstitute naturally occurring deletions that lead to loss of putative silencers located at the 3′ end of the γ-globin genes have been employed, including the 7.2-kb “Corfu” deletion of the γ-δ intergenic region and the 13.6-kb deletion including the γ-δ intergenic region and extending to the first intron of the β-globin gene, similar to the “Sicilian” 12.9-kb HPFH-5 deletion ( Lattanzi et al., 2019 ).

Last but not least, another promising option is base editing by nucleotide deaminases linked to programmable DNA-binding proteins. These proteins function by fusing inactive or nickase Cas9 to deaminases that catalyze the enzymatic conversion of C to T (G-to-A on the opposing strand) or A to G (T-to-C on the opposing strand) ( Gaudelli et al., 2017 ). Because this approach does not involve generation of DNA double strand breaks, it is supposedly safer compared to “classical” genome editing; however, certain limitations exist, as the currently available range of base editors cannot enable conversion of the sickle cell mutation, i.e., direct T-to-A correction. Nevertheless, the strategy can be employed to disrupt alternative sequence elements, analogous to NHEJ-mediated methods, to correct specific mutations of β-thalassemia ( Zeng et al., 2020 ). Subsequent work by Liu and co-workers led to the concept of prime editing, which improved upon the versatility of their base editing tools by inclusion in the RNP particle of a reverse transcriptase and a template for reverse transcription. The resulting tools can precisely introduce all conceivable 12 nucleotide changes as well as small indels ( Anzalone et al., 2019 ). Of note, an extremely interesting study was published in 2016 by Bahal et al. (2016) introducing the use of triplex-forming peptide nucleic acids (PNAs). PNAs are designed in a way that permits their binding to specific genomic DNA sites via strand invasion and formation of PNA/DNA/PNA triplexes (via both Watson–Crick and Hoogsteen binding) with a displaced DNA strand. PNAs are essentially nanoparticles consisting of a charge-neutral peptide-like backbone and nucleobases, enabling hybridization with DNA with high affinity. These PNA/DNA/PNA triplexes are potent in recruiting the cell's endogenous DNA repair systems to initiate site-specific modification of the genome when single-stranded “donor DNAs” are co-delivered as templates containing the desired sequence modifications ( Anzalone et al., 2019 ). The results of this study proved the efficacy of nanoparticles in terms of phenotype correction in the context of monogenic diseases.

Undoubtedly, the research regarding all potential applications in the field of genome editing is very promising and perhaps has better long-term prospects compared to gene therapy by retroviral vectors. Gene addition by designer nucleases outperforms the classical gene addition by retroviral vectors because it provides targeted integration, which, so far, cannot be achieved with retroviral vectors. However, despite potentially higher safety, caveats still exist for genome editing.

The first very important challenge in terms of safety is the identification of the off-target effects. To that end, major efforts have been described including Digenome-seq ( Kim et al., 2015 ) and CIRCLE-seq ( Tsai et al., 2017 ; Lazzarotto et al., 2018 ). Both methods are based on adapter ligation to the CRISPR generated ends: Digenome-seq generates in vitro Cas9-digested whole-genome fragments and then proceeds to profile genome-wide Cas9 off-target effects in human cells. CIRCLE-seq generates a library of circularized genomic DNA with minimized numbers of free ends and subsequent treatment of purified circles with CRISPR/Cas9 RNP complexes followed by adapter ligation and high-throughput sequencing. Although both approaches are highly promising, there are limiting steps such as the length of reads during NGS. Additional efforts such as BLISS ( Yan et al., 2017 ) involve fixation of cells and it is doubtful if there is high accuracy in introducing DSBs as part of the screening (and not the therapeutic) process at high accuracy. Finally the DISCOVER-SEQ ( Wienert et al., 2019 ) approach is based on recruitment of specific DNA repair proteins; hence, it is questionable if all DSBs can be identified, given the fact that even the amount of the engineering agent can have a profound impact on the same cell type: For example, there have been differences described between engineered cord blood CD34 + by lentiviral vectors with low MOI ( Papanikolaou et al., 2015 ) compared to high MOI ( Piras et al., 2017 ). Excluding the actual limitations existing in the current approaches, another point of concern is the fact that some off-targets may be completely benign, whereas others could have serious consequences depending on the cell context or the indication. This is a well-recognized issue in the field and is currently being addressed by engineering the CRISPR payload at both the protein and gRNA level with simultaneous optimization of the ideal window of active exposure of the cells of interest to the functional RNP complex ( Tay et al., 2020 ).

Therefore, the burden from a regulatory aspect is major for the following reasons: (a) Even a single genetic disease caused by knockout of a single gene or sequence may be associated with several mutations, even unrelated ones, in different patients. For example, nobody knows or can accurately predict what can be caused by disruption of the erythroid specific enhancer within the second intron of BCL11A at a population scale. (b) Depending on the indication, even the most well-characterized agents in the field of gene therapy still present surprises. The latest manifestation of tumor generation after lentiviral mediated gene addition in the context of CGD is alarming ( Jofra Hernández et al., 2020 ), as the authors described the development of T cell lymphoblastic lymphoma and myeloid leukemia in 2.94% and 5.88% of the mice tested, respectively, and oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in these mice. (c) Genetic engineering of HSCs presents additional hurdles as CD34 + cells are difficult to be tested for karyotypic analysis, as most of the cells reside in Go phase. This poses a certain challenge toward identification of large chromosomal rearrangements as a result of designer nuclease action in the patients' genome, suggesting the need for development of surrogate assays. For example, approaches introducing chromosomal deletions and not indels will most probably face several difficulties during the transition toward a clinical trial. (d) Last but not least, gene therapy products are often described as “living drugs” and possess totally different pharmacokinetics compared to classical small molecules, and therefore even the regulatory agencies are not streamlined for assessments of such products.

Hence, the transition from bench to the clinic and accordingly for industry toward acquiring marketing authorization will require collaboration between different disciplines including researchers, physicians, industrial stakeholders, regulatory agencies, and policy makers.

Discussion—Future Perspectives

The development of therapeutic approaches based on genome editing by designer nucleases is proceeding with great speed and utilizes as a foundation knowledge produced from decades of traditional gene therapy research. However, any new curative scheme faces new challenges many of which are not foreseen particularly by research labs developing the proof of principle for these important new modalities.

The first perspective under discussion for the entire progress of the field is the actual location at which the therapy will take place. Currently, there are two different models that serve this cause: The centralized model assumes collection of the initial cell product from the patient at a local hospital, shipment of this product to a centralized facility in which the genetic engineering takes place, followed by freezing of the cell therapy product and shipment back to the original location. Thereby, the administering physician thaws the cell engineered product and reinfuses it to the patient. There are several advantages as well as disadvantages with this approach. First, centralized manufacturing is much more familiar with the existing mentality of both regulatory agencies as well as policy makers and governmental or societal stakeholders. However, there are serious limitations: This manufacturing model is intended for products with long shelf life and low degree of personalization, which obviously are not applicable for cell and gene therapy products for which transport can have a profound effect on the underlying biology of the cells of interest. Moreover, there is a high risk of incurring issues related from the distance of the user both geographically and in terms of responsiveness to end user requirements and logistics might face the serious issue of biological waste generation. Generally, the centralized model creates opportunities for errors and mistiming of the cell product delivery.

On the other hand, decentralized manufacturing assumes cell collection and processing locally. Equivalent approaches are currently being employed by hospitals in the context of blood transfusion and transplantation of HSCs. This manufacturing model also has pros and cons: the main advantage of this approach is the general flexibility brought about by being closer to the end user, therefore providing responsiveness to evolving requirements and greater personalization according to patient needs. The area of HSC transplantation has contributed enormously to the progress of the gene therapy field, and from that aspect, the decentralized model is closer to the mentality of tissue transplants, a medical routine since 1975 ( Dunbar et al., 2018 ) and shares a lot of common challenges. However, most of these products are under specific tissue or transplant regulations, and these regulations have debatable applicability on gene therapy products. A key limitation to the decentralized model is exactly one of its assets: the flexibility. For such a manufacturing process to be successful from every possible aspect, it is of critical importance to demonstrate robustness. Therefore, a key question is how it is possible to simultaneously be robust and flexible, specifically taking into account that decentralized manufacturing is based on the expertise and skills of each specialized personnel undertaking the manufacturing in different locations. Another most obvious consideration is the starting material and the variability associated with it. Moreover, the type of culture, the differences in the cultivation media and cytokines used, and the timing of the culture generate additional fluctuations. One plausible approach to decrease user variability or bias would be to apply automation during the manufacturing preferably by closed systems with minimal user interaction. This mentality, ideally could be adopted even from early developments in research labs, suggesting that it would be of great benefit to the field if the cell product was produced already under mock-GMP conditions utilizing automated closed systems and GMP-like grade of media and cytokines. A process of this kind would provide a higher degree of maturity of the cell product and the only open variable step would be the starting material. It should be emphasized that once researchers streamline their processes, they should take into consideration that transfer of a research grade manufacturing to a GMP-like manufacturing would include specific documentation from media and cytokine providers, from retroviral vector providers, and from manufacturers of plasmids or RNPs in the case of genome editing. Also, it is generally advisable to utilize one module in the automation step and not different modules, because the regulatory authorities will ask for specific documentation and accreditation from every single module. Therefore, semi-automation will only create delays during any upcoming evaluation from a regulatory agency compared to full automation. Finally, researchers should keep in mind early on that fetal bovine serum, a material widely used in cell and tissue culture, is not characterized as GMP and therefore it would be eliminated from any future step in the process, requiring optimization of the whole process from the beginning.

As a last remark, successful decentralization would most probably require a new set of highly skilled personnel, possibly creating “technology transfer champions” ( Harrison et al., 2018 ) from the current pool of researchers or students and most importantly students of medical sciences who are young, motivated, and eager to undertake the transition between manufacturing and practice in translational medicine. Additionally, centralized managed control standards and certified operators who receive mandatory re-training and licensing of remote site operations should be seriously considered by the universities, the industry, the government, and the society in general.

Concluding Remarks

The medical field is surely evolving fast and toward the direction of treating diseases previously incurable by the use of genetic manipulations in the form of classical gene therapy by gene addition but also with the advent of designer nucleases by genome editing. Over the past 20 years, significant milestones have been reached in terms of marketing authorization of gene therapy products and real benefit for a large number of patients has been established. However, the field is still in an immature phase, indicating its huge potential for future growth. To that end, researchers should focus early on toward generating true innovative solutions for patients that have the potential to transfer under GMP conditions and are also comparable price wise to the current state of the art. Super expensive solutions, albeit truly innovative in nature, will most certainly face challenges toward achieving proper reimbursement, thereby jeopardizing their eventual availability to patients. It should be emphasized that adoption of poor organization strategies and lack of risk mitigation measures early in the development has the potential to undermine the future success of an otherwise promising strategy or product, specifically in the area of genome editing. If such strategies are adopted early on from researchers, it is possible that previously unforeseen or unanticipated obstacles on the path to approval, often taking decades to address, will be omitted, increasing the wider applicability of genetic therapies, and unlocking their true potential.

Author Contributions

EP researched the literature and wrote the manuscript. AB read the manuscript, provided feedback and final approval. Both authors contributed to the article and approved the submitted version.

Conflict of Interest

AB and EP are employees of Miltenyi Biotec.

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Keywords: genome editing, hemopoietic stem cell, retroviral vectors, designer nucleases, CRISPR

Citation: Papanikolaou E and Bosio A (2021) The Promise and the Hope of Gene Therapy. Front. Genome Ed. 3:618346. doi: 10.3389/fgeed.2021.618346

Received: 16 October 2020; Accepted: 19 January 2021; Published: 24 March 2021.

Reviewed by:

Copyright © 2021 Papanikolaou and Bosio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Eleni Papanikolaou, elinapapanikolaou@gmail.com

This article is part of the Research Topic

Mutation-Specific Gene Editing for Blood Disorders

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Genetic Therapies Benefits and Risks

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In the future, genetic therapies may be used to prevent, treat, or cure certain inherited disorders, such as cystic fibrosis, alpha-1 antitrypsin deficiency, hemophilia, beta thalassemia, and sickle cell disease. They also may be used to treat cancers or infections, including HIV.

Genetic therapies that are currently approved by the FDA are available for people who have  Leber congenital amaurosis , a rare inherited condition that leads to blindness.  CAR T-cell therapy  is FDA approved for people who have blood cancers, such as  acute lymphoblastic leukemia (ALL)  and diffuse large B-cell lymphoma.

Genetic therapies hold promise to treat many diseases, but they are still new approaches to treatment and may have risks. Potential risks could include certain types of cancer, allergic reactions, or damage to organs or tissues if an injection is involved.

Recent advances have made genetic therapies much safer. Better safety has resulted in the FDA approving some gene transfer therapies for clinical use in the United States. There have been a few clinical studies on genome editing, but the approach is much newer than gene transfer. Researchers are still studying the risks.

The National Institutes of Health, which includes the NHLBI, does not perform or fund studies on genome editing targeting sperm, eggs, or embryos in humans. These changes would be passed on to the patient’s children and could have unanticipated effects.

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​Gene Therapy

Gene therapy is a technique that uses a gene(s) to treat, prevent or cure a disease or medical disorder. Often, gene therapy works by adding new copies of a gene that is broken, or by replacing a defective or missing gene in a patient’s cells with a healthy version of that gene. Both inherited genetic diseases (e.g., hemophilia and sickle cell disease) and acquired disorders (e.g., leukemia) have been treated with gene therapy.

Gene therapy. Gene therapy is a direct way to treat genetic conditions as well as other conditions. There are also other related approaches like gene editing. There are many different versions and approaches to gene therapy and gene editing. It all rests on understanding how genes work and how changes in genes can affect our health. Researchers all over the world are studying many different facets of gene therapy and gene editing.

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Gene therapy: principles, challenges and use in clinical practice

• review article
• Open access
• Published: 07 May 2024

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• Cihan Ay MD   ORCID: orcid.org/0000-0003-2607-9717 1 &
• Andreas Reinisch MD PhD 2  

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Introduction

Gene therapy is an emerging topic in medicine. The first products have already been licensed in the European Union for the treatment of immune deficiency, spinal muscular atrophy, hemophilia, retinal dystrophy, a rare neurotransmitter disorder and some hematological cancers, while many more are being assessed in preclinical and clinical trials.

The purpose of this review is to provide an overview of the core principles of gene therapy along with information on challenges and risks. Benefits, adverse effects and potential risks are illustrated based on the examples of hemophilia and spinal muscular atrophy.

At present, in-vitro and in-vivo gene addition or gene augmentation is the most commonly established type of gene therapy. More recently, more sophisticated and precise approaches such as in situ gene editing have moved into focus. However, all types of gene therapy require long-term observation of treated patients to ensure safety, efficacy, predictability and durability. Important safety concerns include immune reactions to the vector, the foreign DNA or the new protein resulting from gene therapy, and a remaining low cancer risk based on insertional mutagenesis. Ethical and regulatory issues need to be addressed, and new reimbursement models are called for to ease the financial burden that this new treatment poses for the health care system.

Gene therapy holds great promise for considerable improvement or even cure of genetic diseases with serious clinical consequences. However, a number of questions and issues need to be clarified to ensure broad accessibility of safe and efficacious products.

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Already in 1972, Friedmann and Roblin hypothesized that genetic modification might be the way to cure hereditary diseases [ 1 ]. Following many years of scientific groundwork and technical advancements, the first clinical gene therapy studies started in the early 1990s [ 2 ]. Over the years, several major setbacks, including the tragic death of a patient treated in a gene therapy trial in 1999 and several cases of unintended insertional mutagenesis and development of acute leukemia, slowed the development [ 3 , 4 , 5 , 6 , 7 ]. The 18-year-old patient who died in 1999 had partial ornithine transcarbamoylase (OTC) deficiency, a genetically determined metabolic disorder of the urea cycle, and received an infusion of corrective OTC gene encased in a recombinant adenoviral vector [ 8 ]. A severe immune reaction evoked by the adenoviral vector led to his death four days after the administration. This case highlighted the potential of the vector itself to pose a risk, adequate training of the health care staff and the implementation of basic operating procedures, among others.

The lessons learned from these events enabled continuous improvements, and the unprecedented results that are achievable with gene therapy led to the development of a myriad of new products currently tested in clinical trials for a broad range of indications [ 9 ]. Today, gene therapies have emerged as promising treatment options and are rapidly entering the treatment landscape of various inherited and acquired diseases including immune disorders, neurodegenerative diseases, hemophilia, ocular diseases, hemoglobinopathies, or cancer. Some gene therapies have already been approved for clinical use, and many more are being developed at increasing speed.

Although continuous collection of additional long-term safety data will be necessary in the future, the growing importance of gene therapy is beyond doubt and calls for appropriate knowledge among health care professionals. There is currently a high unmet need for education, as revealed by a survey conducted among hospital physicians in Austria [ 10 ]. To address this knowledge gap, this review summarizes core principles, benefits, potential risks and challenges of gene therapy, with a particular focus on hemophilia and spinal muscular atrophy, and discusses future perspectives.

Basic principles of gene therapy

Methods and techniques of gene therapy: gene addition/augmentation vs. gene suppression.

Gene therapy is the transfer of genetic material to a patient to treat or potentially even cure a disease. There are various approaches of gene therapy. Most currently used gene therapy products attempt to replace the function of a defective gene with that of a healthy gene. The genetic material (aka transgene) should ideally be delivered to the physiologically relevant target tissue where it is expressed at a physiologically meaningful level and in a stable manner. Interference of the gene or its protein products with the integrity of the target cells must be avoided [ 11 ].

This addition or substitution of genes with loss-of-function defects (Fig.  1 a) is called gene addition/augmentation [ 2 ]. In case of gene augmentation, the newly transferred functional copy of a gene is present in the cell nucleus together with the defective gene.

Types of gene therapy: gene augmentation, gene suppression, and genome editing (according to Anguela XM and High KA, and Porteus MH) [ 2 , 12 ]. Foot note: NHEJ  non-homologous end joining; repair of double-strand breaks via direct ligation of the break ends without a homologous template

A different approach is required when the disease is caused by gain-of-function defects. Suppression of gain of function can be achieved by the import of inhibitory sequences (i.e., microRNAs, short hairpin RNAs) into target cells (Fig.  1 b).

Genome editing

More recently, with the discovery of novel tools that can precisely target and manipulate DNA, in situ repair of genetic defects has become possible. This approach is called genome editing and allows for the correction of genetic defects with single base-pair precision (Fig.  1 c; [ 2 , 12 ]). Genome editing mostly relies on the site-specific introduction of a double-strand break (DSB) into DNA. The nuclease-induced site-specific DSB in the genome stimulates active endogenous repair mechanisms. The two best understood repair mechanisms are non-homologous end-joining (NHEJ) and homology-directed repair (HDR). In NHEJ, the DSB is fixed simply by joining the two ends of the broken DNA. This mechanism is relatively error-prone and frequently leads to insertions and deletions (indels) that can result in the functional loss of a gene. In stark contrast, HDR—the naturally occurring recombination mechanisms observed in mammalian cells—involves the use of the sister chromatide as a template for a “copy and paste” process known as homologous recombination. However, for genome editing, a cell can be tricked, and a DNA donor repair template can be introduced into the cell that will be used for homologous recombination instead of the sister chromatide. Importantly, this DNA repair template can be engineered to correct a mutation or even integrate additional genetic material.

CRISPR-Cas9 is currently the platform that is used most frequently for the introduction of DNA DSBs. However, other nucleases such as zinc-finger nucleases, transcription activator-like effector nucleases and homing endonucleases-meganucleases offer comparable precision.

Genome editing can be performed in cells both outside and inside of the body ( ex vivo and in vivo , see below) [ 12 ]. Clinical trials are currently assessing the utility of genome editing in the correction of monogenic diseases and cell-based regenerative medicine; also, enhancement of chimeric antigen receptor (CAR) T cell therapy is investigated. In principle, genome editing has the potential to halt the progression of most monogenic diseases, provided that patients are treated before irreversible damage has occurred. The first CRISPR-based gene therapy, which has been approved only recently in the European Union is exagamglogene autotemcel for the treatment of transfusion-dependent β‑thalassemia and severe sickle cell disease [ 13 ].

Methods of gene transfer: ex vivo vs. in vivo gene therapy

Ex vivo gene therapy requires the extraction of cells from the patient [ 14 ]. After the successful introduction of a transgene or successful gene editing, the cells are re-administered to the patient. Ex vivo gene therapies commonly use cells of hematopoietic origin such as hematopoietic stem cells or T cells, and are being developed for the treatment of inherited immune disorders, neurodegenerative diseases (e.g., X‑linked adrenoleukodystrophy, metachromatic leukodystrophy), β‑hemoglobinopathies (e.g., β‑thalassemia, sickle cell disease), and cancer.

Today, gene therapy for cancer has mainly been established in the form of CAR T cell therapy. CARs endow T cells with the ability to target antigens expressed on the surface of tumor cells. CD19, an antigen present in most B cell malignancies, constitutes the classical target, with several products having been approved to date. However, CAR T cell applications are extending to other hematologic malignancies as well as solid tumors.

In contrast to ex vivo gene therapy, in vivo gene therapy relies on the administration of a vector that carries and delivers the transgene to a target tissue (e.g., liver, neurons, muscle). Most current in vivo gene therapies target the patient’s liver and can be applied intravenously [ 15 ]. In vivo gene therapy usually does not require conditioning prior to administration; thus, prolonged hospital stays are avoided, and the treatment can frequently even be conducted in the outpatient setting. Moreover, this approach is attractive as it dispenses with the need for elaborate steps involved in ex vivo treatment including cell collection from the patient and manipulation in a specialized facility before re-administration [ 16 ]. However, the feasibility of in vivo administration depends on tissue-specific targeting or local delivery and/or target-cell-specific gene expression.

In vivo gene therapy primarily focuses on rare monogenic disorders caused by loss-of-function or toxic gain-of-function mutations. Among others, in vivo gene therapy is currently being evaluated or has already been approved in the treatment of hemophilia A and B, neuromuscular disorders (e.g., spinal muscular atrophy, Duchenne muscular dystrophy) and various types of inherited blindness (e.g., RPE65 mutation-associated retinal dystrophy, achromatopsia, choroideremia, Leber’s hereditary optic neuropathy, X‑linked retinoschisis, and X-linked retinitis pigmentosa). In the setting of central nervous system diseases, targeting a sufficient number of cells to achieve an adequate level of gene modification is challenging [ 2 ].

Vectors are vehicles that can carry genetic material and introduce it into target cells. For gene transfer, mainly naturally occurring viruses are genetically modified for the purpose of transferring and expressing a transgene. In viral vectors, the viral genome is replaced by the gene therapy transgene. One fundamental distinction between the viruses used for gene therapy is their inherent capacity to integrate into the host DNA. Therefore, viral vectors can broadly be classified as either integrating or non-integrating [ 17 , 18 ].

Integrating viral vectors are introduced into cells with the aim of stably incorporating therapeutic genes into the genome, thus allowing the cells to pass the transgene onto every daughter cell. These vectors, which are typically derived from retro- and lentiviruses, are frequently employed for ex vivo gene therapy.

With non-integrating viral vectors, on the other hand, the transferred DNA is stabilized extrachromosomally as an episome. Since the transgene is usually not integrated into the genome, it needs to be delivered to long-lived, non-dividing, post-mitotic cells where it will be expressed for the life of the target cell only. Episomes are stable in non-dividing cells for long periods and provide sustained transgene expression [ 19 , 20 ]. On the downside, transgene expression may be lost over time upon cell proliferation due to the lack of vector genome replication with cell division [ 21 ]. Non-integrating vectors are typically used for in vivo gene therapy.

Recombinant adeno-associated viral (rAAV) vectors have emerged as the platform of choice for in vivo gene therapies due to their advantages of relatively low immunogenicity, targeted gene delivery into a range of tissues, and long-term expression of the transgene [ 22 , 23 , 24 ]. AAV is a very small ( parvovirus) single-stranded DNA virus that is non-pathogenic and naturally replication-defective. Wild-type AAV requires the presence of another virus, such as an adenovirus, to replicate [ 25 ]. In the process of engineering, all viral coding sequences including the rep and cap genes that are responsible for replication and the structure of the viral capsid are replaced with a gene expression cassette of interest. This includes not only the therapeutic gene but also other transcriptional regulatory elements such as a promoter sequence that facilitates transgene expression within specific cell types [ 26 ]. rAAV vectors have tropism for specific tissues depending on their serotype, with serotypes ranging from AAV1 to AAV13 [ 27 ]. Features of different vectors are discussed in Table  1 [ 2 , 28 ].

For the large-scale production of rAAV vectors, platforms based on human embryonic kidney cells (HEK) or the insect cell line Spodoptera frugiperda (SF9) with recombinant baculoviruses have been widely employed [ 22 ]. rAAVs are administered via a single infusion either intravenously or locally. If given intravenously, the vector will transduce the target cell depending on its tissue tropism. Once bound to the cell via receptors, the virus gets endocytosed. After escaping from the endosome, rAAV particles enter the cell nucleus, the viral capsid gets uncoated, and after a second strand synthesis of the transgene, the host cell’s endogenous transcription and translation machinery is used for the production of a functional protein. Based on the fact that rAAVs integrate into the host genome at very low frequencies, rAAV is considered to bear only low risk of genotoxicity [ 29 , 30 , 31 , 32 , 33 ]. In clinical studies investigating valoctocogene roxaparvovec and etranacogene dezaparvovec that have been licensed for the treatment of hemophilia A and B, respectively, transgene DNA was temporarily detected in semen; therefore, barrier contraception is recommended for 6 and 12 months after the administration of valoctocogene roxaparvovec and etranacogene dezaparvovec, respectively, in patients with reproductive potential [ 34 , 35 ]. Moreover, treated patients should not donate semen, blood, organs, tissues or cells for transplantation.

Risks of gene therapy

Depending on the type of gene therapy ( ex vivo vs. in vivo , integrating vs. non-integrating vectors), several safety-related issues need to be taken into consideration and should be discussed with the potential patient.

Integrating vectors such as retro- and lentiviruses that are primarily used for ex vivo gene therapy bear the risk of insertional mutagenesis due to their semi-random integration into the DNA. This can potentially induce the activation of an oncogene or the disruption of a tumor suppressor gene, thereby leading to the formation of cancer [ 6 , 36 , 37 , 38 ]. Unfortunately, T cell leukemia developed in some of the early trials using γ‑retroviral vectors for severe combined immunodeficiencies (SCID) [ 39 ]. Over time, the risk of insertional mutagenesis has been reduced by the development of safer vectors [ 18 ]. Compared to γ‑retroviral vectors, lentiviral vectors have a safer integration pattern and higher transduction efficiencies. However, clinical-scale production of lentiviral vectors is challenging. Nevertheless, specific surveillance and long-term follow-up is necessary. In the future, such unintentional detrimental integration events might be avoided by using the very precise genome editing technology [ 18 ].

In contrast to integrating viral vectors that are primarily used for ex vivo gene therapy applications, non-integrating viral vectors are mainly used for in vivo gene therapy. These have only minimal rates of integration into the donor DNA and consequently confer a very low probability of causing insertional mutagenesis and cancer. Hemophilic dogs treated with AAV gene therapy had low but detectable levels of AAV integration into the genomic DNA and did not show any evidence of tumor formation after 10 years of follow-up [ 40 ]. Studies in neonatal mice implicated that pathogenic AAV integration events might actively contribute to hepatocellular cancer development, although potential genotoxic events are highly dependent on factors including AAV integration preferences, vector design, vector dose and, in particular, recipient age at AAV injection [ 41 , 42 , 43 ].

Since non-integrating vectors are applied in vivo , they carry the risk of evoking immune responses that are potentially life-threatening or might impair the long-term efficacy of treatment. Immune responses and related adverse events seem to be directly associated with the vector doses applied [ 44 , 45 ]. Uncontrolled immune responses are the main culprit with regard to most severe adverse events linked to AAV gene transfer, including fatal hepatotoxicity, dorsal root ganglia toxicity, and myocarditis. Notably, the human body contains immune-privileged sites (e.g., the central nervous system) and immunosuppressive microenvironments (e.g., the liver) where AAV vectors are less likely to trigger strong responses than at other sites such as the circulation or the muscle [ 46 ].

Uncontrolled innate immunes responses such as overactivation of the complement pathway with subsequent induction of thrombotic microangiopathy have been described following AAV gene therapy. Thrombotic microangiopathy is a hematologic emergency situation caused by microscopic blood clots in the capillaries and small blood vessels, leading to organ damage, anemia and low platelet counts [ 47 ]. Also, the adaptive immune system can cause dangerous side effects via CD8+ cytotoxic T‑cell responses, such as T‑cell mediated hepatotoxicities associated with inflammatory reactions that have been observed in AAV9 vector therapy for spinal muscular atrophy.

Immune responses to vectors can be mitigated by the administration of immunomodulatory drugs such as corticosteroids [ 18 ]. However, immune system-mediated toxicity is still a challenge for successful gene transfer using AAV vectors, particularly in settings in which the treatment of the targeted genetic disease requires high doses [ 48 ].

Approved therapies and fields of investigation

A number of gene therapy products have been licensed over the last seven years in Europe, the United States and other countries. Currently, a total of six CAR T cell products have received approval in Europe. Tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel and lisocabtagene maraleucel are used for the treatment of patients with B‑cell malignancies (e.g., lymphoma); all of these target the CD19 antigen [ 49 , 50 , 51 , 52 ]. Tisagenlecleucel is also indicated for acute lymphoblastic leukemia [ 49 ]. The BCMA-directed therapies idecabtagene vicleucel and ciltacabtagene autoleucel have been licensed for the treatment of multiple myeloma [ 53 , 54 ]. Talimogene laherparepvec is a modified oncolytic herpes virus that is used as an intralesional cancer immunotherapy for advanced melanoma [ 55 ].

In addition, at the time of the publication of this review, gene therapies are available in Europe for serious monogenic disorders including severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID; autologous CD34+ cells transduced with a retroviral vector that encodes for the human ADA complementary DNA sequence), biallelic RPE65 mutation-associated retinal dystrophy (voretigene neparvovec), aromatic L‑amino acid decarboxylase (AADC) deficiency (eladocagene exuparvovec), metachromatic leukodystrophy (atidarsagene autotemcel), spinal muscular atrophy (onasemnogene abeparvovec), hemophilia A (valoctocogene roxaparvovec) and B (etranacogene dezaparvovec) and β‑thalassemia as well as sickle cell disease [ 13 , 34 , 35 , 56 , 57 , 58 , 59 , 60 ].

A multitude of trial programs is currently evaluating gene therapies in a broad range of diseases. Approximately 1500 products are being tested in the pre-clinical setting and in more than 500 clinical studies. In addition to the mentioned indications, gene therapy is being assessed in inherited metabolic diseases such as ornithine transcarbamylase deficiency (NCT02991144), homozygous familial hypercholesterolemia (NCT02651675) and mucopolysaccharidosis type VI (NCT03173521), in age-related macular degeneration (NCT01024998, NCT01301443, NCT01494805, NCT03066258) and previously untreatable disorders like Huntington’s disease (NCT03761849), among many others. As an example, achievements and limitations of established gene therapies are delineated below for hemophilia and spinal muscular atrophy.

Hemophilia a and b

Hemophilia, an X‑linked recessive bleeding disorder, is caused by a deficiency of coagulation factor VIII (hemophilia A) or IX (hemophilia B) due to mutations in the genes encoding for these factors. Several characteristics make hemophilia A and B an ideal target for gene therapy: this is a monogenic, recessive disease which results in a large range of affected protein levels [ 61 , 62 , 63 ]. Moreover, the bleeding phenotype is responsive to increases of factor levels, and their measurement provides monitoring of the treatment efficacy. While FVIII is synthesized in the sinusoidal endothelial cells of the liver, FIX synthesis takes place in the hepatocytes [ 63 , 64 ]. The majority of defects of the F8 gene are caused by intron 22 inversions; in the F9 gene, missense mutations are mainly responsible for the absence or dysfunction of the clotting factor [ 65 , 66 ].

In patients with hemophilia, FVIII or FIX deficiency leads to bleeding into joints, muscles and soft tissues, eventually giving rise to joint damage, disability and chronic pain as the most common consequences [ 61 ]. The traditional treatment consists of intravenous replacement of coagulation factor concentrates at regular intervals, given the relatively short half-life of these factors. This puts a considerable burden on patients and care givers. Furthermore, persons with hemophilia may develop inhibitory antibodies that diminish the efficacy of factor replacement. Despite regular prophylaxis, the risk of arthropathy is not completely reduced with the current treatment options. Moreover, the treatment confers a significant cost burden, and access to factor products is limited in many countries.

Valoctocogene roxaparvovec was the first gene therapy to be licensed for the treatment of hemophilia A and became available in August 2022 in the European Union [ 34 ]. Similarly, etranacogene dezaparvovec was approved as the first gene therapy for hemophilia B in February 2023 [ 35 ].

Gene therapy for hemophilia is liver-directed as the vectors target hepatocytes, which act as protein factories to release the transgene product into the circulation. AAV vectors with the serotype 5 are used for both currently approved liver-directed therapies. This treatment is expected to transform severe disease phenotypes into mild or normal phenotypes based on sustained elevation of clotting factor levels [ 2 , 63 , 67 , 68 ]. The continuous expression of coagulation factors provides protection from bleeding, renders prophylaxis at regular intervals unnecessary and contributes to increased quality of life.

While hemophilia A and B show similar clinical symptoms, their molecular bases differ. As FVIII complementary DNA is larger than FIX complementary DNA (approximately 9 kb vs. 1.5 kb), modification is required to enable packaging of the F8 transgene into the recombinant AAV5 (rAAV5) vector [ 65 , 66 , 69 ] that has limited packaging capacity of approximately 4.7 kb (Fig.  2 ). To fit the F8 transgene into AAV, the large B‑domain of F8 is deleted, resulting in a length of approximately 5 kb. For the F9 transgene, a naturally occurring but more active variant of FIX that was initially described in a family in Padua (i.e., the Padua variant) is often used [ 70 ]. The therapy is administered as a single intravenous infusion, with dosing based on body weight. Following the administration, patients may develop a mild viral syndrome consisting of transient fever, myalgia, and malaise [ 71 , 72 , 73 ].

Structure of adeno-associated viral vectors for the treatment of hemophilia ( a ) and ( b ). Foot note: ITR  inverted terminal repeat; pA  polyadenylation signal

AAVs naturally infect humans, and upon infection the human immune system develops neutralizing antibodies that are a particular challenge for AAV-based gene therapy approaches. These pre-existing neutralizing anti-AAV antibodies impede gene transfer by inhibiting the transduction of target cells by the AAV-based vector [ 74 ]. Measurable antibodies to different AAV serotypes have been found in approximately 30–60% of the population [ 75 ]. Prior to treatment with the gene therapy product approved for hemophilia, the levels of neutralizing antibodies need to be assessed. Only patients without antibodies according to a validated assay are eligible for the administration of valoctocogene roxaparvovec [ 34 ]. With respect to etranacogene dezaparvovec, patients with pre-existing anti-AAV5 antibodies were not excluded from the phase III trial. Trials results showed that gene therapy can be successful even in the presence of low titers of pre-existing neutralizing antibodies; however, the titer should not exceed 1:678 according to the specific assay employed for etranacogene dezaparvovec [ 35 ].

Accurate and robust detection of neutralizing anti-AAV antibodies is important but not easy to achieve as the required assays have not been established in clinical routine yet. Furthermore, no universal method has been implemented to reliably measure the amount of clinically relevant antibody levels [ 76 ]. Transduction inhibition assays and total antibody assays are used, although meaningful comparisons across assays are nearly impossible due to the lack of standardization. The limited availability of head-to-head studies that align assay results with clinical outcomes renders the interpretation and implementation of screening titer cutoffs difficult [ 77 ].

Another issue that requires attention is the emergence of potential immune responses against capsid proteins or even the transgene and its products that can lead to rejection of the transduced cells [ 78 , 79 , 80 ]. In a high number of patients, liver-directed gene therapy for hemophilia led to modest increases in the liver transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST) [ 78 ]. Although all hemophilia gene therapy clinical trials have shown transaminitis, this was more frequently seen in patients receiving hemophilia A gene therapy than in those undergoing hemophilia B gene therapy [ 81 , 82 ]. In the majority of cases, the reported elevations in ALT levels showed a 1.5- to 2‑fold peak above the upper limit of normal. Unfortunately, the mechanisms responsible for ALT elevation potentially reflecting liver damage have not been fully unraveled to date, but cytotoxic T‑cell attacks against transduced cells and/or cellular stress induced by the accumulation of misfolded protein in the endoplasmic reticulum are suspected [ 20 , 23 , 83 ]. Transaminitis mainly occurred within the first 12 weeks after vector infusion and either preceded a loss of transgene expression or coincided with it [ 78 , 84 ]. In clinical studies, immunosuppression with corticosteroids was initiated with the aim of dampening the immune system and thereby preserving the expression of the gene therapy product [ 80 ].

During the first weeks and months following administration of gene therapy, close clinical and laboratory monitoring is mandatory [ 66 ]. If the gene transfer is successful, the need for exogenous administration of coagulation factor products generally declines considerably until the endogenous factor production has sufficiently increased to render factor replacement therapy unnecessary [ 85 , 86 ]. However, patients need to be aware of considerable inter-patient outcome variability that has been observed in clinical trials. Additionally, each gene therapy product has its unique features, including vector design and vector dose, AAV serotype and the production platform used for manufacturing. Patient variables include previous AAV exposure, patient-specific antigen processing, and hepatic health prior to gene therapy [ 44 , 71 , 85 , 87 , 88 , 89 ]. Further research and long-term observation is needed to gain additional insights, especially with respect to safety as well as predictability and durability of factor expression.

• Spinal muscular atrophy

Loss-of-function mutations in the survival motor neuron 1 ( SMN1 ) gene give rise to spinal muscular atrophy (SMA) [ 90 ]. This autosomal recessive disease is characterized by the degeneration of alpha motor neurons located in the spinal cord. Progressive muscle weakness, paralysis, loss of bulbar function and death from respiratory complications occur at around 2 years of age in most patients [ 91 , 92 ]. Infantile-onset (type 1) SMA is the most severe and most common subtype of SMA [ 93 ]. It usually manifests before the age of 6 months and is the most common genetic cause of death in infants; however, symptoms may already be present at birth.

The antisense oligonucleotide drug nusinersen has revolutionized the treatment of patients with SMA. It targets the SMN2 gene, which is a nearly identical copy of the SMN1 gene, and produces functional SMN protein, although only a fraction of the amount obtained from the intact SMN1 gene, and thus cannot compensate for the loss of SMN1 [ 94 , 95 ]. By modulating alternative mRNA splicing of the SMN2 gene in spinal motor neurons, nusinersen induces higher expression of SMN2 , thereby better compensating for the SMN1 loss. However, this treatment involves repeated intrathecal administration (i.e., direct injection into cerebrospinal fluid) with up to seven injections during the first year followed by maintenance doses every 4 months. The treatment costs are substantial, and patients with advanced disease still rely on assisted respiration using non-invasive ventilation [ 96 ]. In addition, the oral SMN2 pre-mRNA splicing modifier risdiplam has been approved for the treatment of patients with 5q-autosomal recessive SMA with a clinical diagnosis of SMA types 1, 2, or 3, or with one to four copies of the SMN2 gene [ 97 ].

The first gene therapy for patients with SMA is onasemnogene abeparvovec, which was approved in the European Union in 2020. It is indicated in patients with SMA linked to chromosome 5q, a biallelic mutation in the SMN1 gene and clinically apparent SMA type 1, or 5q-associated SMA with a biallelic mutation in the SMN1 gene and up to three copies of the SMN2 gene [ 60 ]. Onasemnogene abeparvovec is an AAV-based gene therapy that is administered as a one-time intravenous infusion. The AAV vector serotype 9 (AAV9) delivers a fully functional copy of the SMN gene into the target motor neuron cells, leading to expression of the SMN protein.

SMA patients treated with onasemnogene abeparvovec show improvements in muscle movement and function, significant improvement in their ability to reach developmental motor milestones, and survival prolongation. Long-term study results suggest evidence of sustained clinical efficacy. The phase I START study included symptomatic infants with SMA type 1 and two copies of the SMN2 gene [ 98 ]. After a median of 5.2 years post gene therapy, all 10 patients in the therapeutic-dose cohort remained alive and without the need for permanent ventilation. All of them had maintained previously acquired motor milestones, and two had achieved the milestone of standing without assistance. The phase III SPR1NT trial evaluated onasemnogene abeparvovec in pre-symptomatic children with biallelic SMN1 mutations treated within 6 weeks after birth [ 99 ]. Among 15 children with three SMN2 copies, all were able to stand independently before 24 months, and 14 walked independently. All of them survived without permanent ventilation at 14 months.

Mandatory assessments prior to the treatment include measurement of pre-existing AAV9 antibodies using a validated assay and liver function tests. The most common side effects of onasemnogene abeparvovec comprise elevation of liver enzymes and vomiting. As acute hepatic failure with a fatal outcome has been reported, it is recommended to monitor the liver function regularly for at least 3 months after treatment [ 100 ]. Immune responses to the vector are assumed to be the cause of hepatotoxicity; therefore, a prophylactic corticosteroid regimen needs to be administered. Moreover, available data suggest that overexpression of the SMN protein, especially in the sensorimotor circuit, might lead to gain of toxic function [ 101 ]. A long-term follow-up study (NCT03421977) of the completed phase 1 study (NCT02122952) is assessing safety for up to 15 years, with final results expected for December 2033.

Challenges & perspectives

Gene therapy has opened new doors in the treatment of a range of serious and debilitating diseases. However, many remaining challenges need to be fully addressed before gene therapy can become a routine treatment for monogenic diseases [ 18 , 102 ]. These include mainly aspects related to safety, predictability, and the durability of the gene therapy outcomes. For in vivo gene therapy, better understanding of immune responses is needed, and systematic long-term efficacy and safety assessment of every treated patient will be essential. Moreover, manufacturing and regulatory challenges need to be solved to make gene therapies broadly accessible. Since gene therapy requires well-trained personnel working at specialized facilities, the number of centers providing these therapies will be limited [ 2 ].

Finally, a societal consensus needs to be reached regarding disputed issues such as the very high financial burden [ 18 ]. One-time gene therapies tend to be extremely expensive up-front, although cost-benefit analyses that take patient quality of life and lifelong medical costs of currently available treatments into account may provide justification for the use of gene therapy products [ 103 ]. In addition, treatment options have been completely absent for a range of serious diseases to date. Nevertheless, keeping the expenses at a reasonable level will be important to improve equality of access. Dedicated funding programs can help to lower the financial burden. Negotiations with health insurances and government agencies might result in the development of new models for reimbursement.

Finally, to implement gene therapy in clinical practice special logistics and a multidisciplinary approach will be required. Various organization in the field of hemophilia propose new delivery models, such as the hub and spoke model to gain access to gene therapy for patients (summarized in [ 104 ]). In such a care delivery model a close collaboration and communication between the hub center, which is responsible for administration of gene therapy and the spoke center (i.e. the referral center) is needed to cover the management of the complex patient journey form initial discussion to long-term follow up.

Gene therapies are promising and offer enormous potential with respect to finally achieving cure of many serious hereditary and non-hereditary diseases. In 2024, the history of their development already spans decades, although in clinical terms, it appears that the journey has barely begun. Study results obtained with approved gene therapies have proven the principle of gene therapy for clinical use. Nevertheless, to make this new treatment approach broadly available, very demanding challenges regarding both medical and regulatory/financial issues need to be addressed. Long-term safety, clinical efficacy and advantages over standard treatment options must be clearly demonstrated to justify the high-cost burden. Also, ethical discussions are needed to determine an acceptable framework for these new procedures. With numerous trials investigating gene therapies in various indications ongoing, patients with devastating diseases can now hope for new and unprecedented treatment options.

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Summary of Product Characteristics Yescarta®.

Summary of Product Characteristics Tecartus®.

Summary of Product Characteristics Breyanzi®.

Summary of Product Characteristics Abecma®.

Summary of Product Characteristics Carvykti®.

Summary of Product Characteristics Imlygic®.

Summary of Product Characteristics Strimvelis®.

Summary of Product Characteristics Luxturna®.

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Summary of Product Characteristics Libmeldy®.

Summary of Product Characteristics Zolgensma®.

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The authors thank Dr. Judith Moser for providing assistance in medical writing and editorial support, which was funded by Pfizer Corporation Austria Ges.m.b.H.

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Gene Therapy: Risks and Benefits Research Paper

Introduction, summary of the mechanisms of gene therapy, discussion of paper and critique of the methods used, argument and future experiments proposed, works cited.

Gene therapy is the “insertion or removal of genes which can also be alternated within the cell or tissues of an organism for purposes of treating diseases” (Cross & Burmester). All over the world, “the technique is best known for the correction of defective genes so as to treat diseases; the most common procedural form of gene therapy involves the insertion of the functional gene in order to replace the mutated gene within an organism” (Cross & Burmester).

Over the recent past use of gene therapy has revolutionized the treatment approaches, and research on this subject is justified because of the great potential that this technique offers. Similarly, further research will also shed light on possible risk factors that are associated with this method of treatment. The purpose of this paper is to undertake a cost-benefit analysis of gene therapy as a treatment option.

Germ-line gene therapy

Under normal circumstances, germ-line gene therapy procedures involve alternating and replacing all defective genes within the body of an organism (Walesgenepark.co.uk). In this case, functional genes are isolated and inserted into the interior lobes of the reproductive tissues or cells of the relevant organism. Considerably, this therapy operates on the basis that fresh and healthy genes get inoculated into the gametes of an organism in order to reduce the risks of later transfer of the defective genes to the next generation of the organism (Walesgenepark.co.uk).

Moreover, there is the involvement of complete alteration of the genetic makeup of early-stage blastomere so as to enhance changes of the genetic codes which can be passed on from generation to generation. Secondly, germ-line therapy also involves alteration of the gametes before fertilization since, after fertilization, the characteristics are passed on to the offspring’s (Walesgenepark.co.uk).

Somatic gene therapy

Generally, “somatic gene therapy involves the process of alteration of the genetic makeup of somatic cells (i.e., all body cells except sex cells) of an individual” (Rothchild, Laura & Lauren). Contrary to germ-line therapy, there is no transfer of these cellular changes to the next generation of the organism, simply because they are neither sex cells nor gametes which fuse during fertilization (Rothchild, Laura & Lauren). The main focus on the process involves changing the arrangements of the genetic codes of the specific cells using either an in-vitro or ex-vivo DNA delivery system. This technique in today’s life can be employed medically in treatments of a variety of diseases, including; hemophilia, muscular dystrophy, among many others; currently, it is used for cancer treatments (Rothchild et al.).

Telomerase inhibition strategies

In humans, it’s evident that the telomerase RNA abbreviated (hTR) plays an important role in anticancer therapy. This hTR can be used as an anticancer either individually or in combination with another human telomerase reverse transcriptase (hTERT) (Li, Li, Yao, Geng, Xie, Feng, Zhang, Kong, Xue, Cheng, Zhou & Xiao 4). Current findings indicate that when these two agents combine with the recombinant adenovirus, another nucleotide called small-interfering RNA (siRNA) is formed (Li et al. 4).

Furthermore, it has been established that the levels of telomerase activities together with mRNA, hTR are greatly reduced by the activities of the recombinant adenovirus resulting in inhibition of Xenograft tumor growth (Li et al. 4). This implies that siRNA, which is specifically expressed in recombinant adenoviruses, is the best tool to be used as an anticancer and also in the treatment of oral squamous cell carcinoma (OSCC) (Li et al. 4). The major advantage of this technique is that the anticancer effect on OSCC is virtually accomplished by cellular proliferation in addition to cellular apoptosis (inhibition of tumor angiogenesis) (Li et al. 4).

Monoclonal antibodies in target therapies of breast cancer

Globally, breast cancer is known to be a killer disease that largely affects females; the major causative agent in about 10% of world breast cancer cases is the mutation of the gene, which is inherited from any of the parents (Grammatikakis, Zervoudis & Kassanos 640). Furthermore, the most effective known therapeutic alternative diagnosis or treatment of breast cancer globally is the use of gene therapy. Some of the procedures used in treatment include molecular chemotherapy, antiangiogenic gene therapy, among many other therapies currently being used (Grammatikakis et al. 640).

bacteria-mediated anti-angiogenesis therapy

Most of the recent studies have revealed that there are some bacterial species that are capable of colonizing solid tumors. This inherited characteristic, however, can further be enhanced via genetic engineering, developing a natural anti-tumor activity that always enables the specific bacteria to transfer its therapeutic molecules directly into the target cells (Gardlik, Behuliak, Palffy, Celec & Li 7). There are few completed studies that have completely documented the anti-angiogenesis process, which is basically a bacterial mediated therapy for cancer (Gardlik et al. 7).

There are four recognized approaches that scientists use when utilizing the use of bacteria in cancer treatments; these include anticancer therapeutic-autofiction, DNA vaccination, transkingdom RNA interference, and alternative gene therapy (Gardlik et al. 7). Notable to mention is that the major primary goal of all these approaches is that they all focus on stimulation of angiogenesis suppression.

Based on the evidence of this paper, the discussion focuses on the argument of whether gene therapy is effective when the cost-benefit analysis is undertaken. Personally, I totally agree with the essay topic that gene therapy has more benefits than costs since it has been successful in the treatment of most chronic cancerous diseases. Gene therapy usually works by relying on immunotherapy and the use of vector organisms like viral particles to modify the genome of the host cell that triggers an immune response, which finally destroys the cancer cells present in the body (Cross & Burmester).

By using gene therapy, many cancerous diseases, i.e., lung, prostate, pancreatic cancers, have a higher chance of being completely treated. As such, gene therapy is now emerging to be the most common preferred treatment of choice because of its efficacy over other treatment methods (Cross & Burmester). The gene treatment also allows the use of a single vector or a combination of several vectors aimed at achieving optimal results (Cross & Burmester).

Currently, further research on gene therapy is still ongoing, and various clinical trials have so far been completed, which now has led to the evolution of vaccine productions (Cross & Burmester). The vaccines are promising to be the most effective technique since they only require autologous cells to have them manufactured, although this has many cost implications; this is one of the disadvantages of this technique. The second disadvantage is that very few hospitals globally are capable of having such vaccines manufactured because of the costly technology associated with the production of the vaccine. All these factors ultimately limit the availability of the vaccine as a viable treatment option.

A recent research study provides findings that show evidence of secondary gliomas stem cell, which originates from an astrocytic tumor that contains a genetic mutation that possesses the tumor suppressor gene recognized as p53 (Cross & Burmester). As part of the procedure, it was proposed in the experiments that in order to induce apoptosis of tumor cells, one must incorporate the use of an integrated suicide factor together with the adenovirus-mediated transfer of p53 (Cross & Burmester). Considerably, the main strength of this approach is supported by the fact that p53 mediate apoptosis follows two distinct and separate pathways reducing pathogenicity (Cross & Burmester).

Gene therapy technology has brought a great 21st-century revolution to the modern system of disease treatments, precisely when it comes to cancer treatments. It is remarkable to note that the development of anticancer treatments by modified immunotherapy and gene therapy, among others, has helped to cure many cases of cancers and saved many others from death. However, through gene therapy, many victims of cancer have attained a prolonged lifespan after being subjected to this treatment.

Scientifically, the principle behind gene therapy when it comes to cancer treatment is that a successful cancer treatment involving therapeutic modality always aims at real activation of death pathways within the cancer cells.

Therefore, there is no doubt that for cancerous diseases, the development of genetic engineering and specific cancer vaccines are proving to be the most effective treatment approaches. There are hope and confidence that in the near future, all obstacles encountered during the first generation cancerous and precancerous treatments will be eliminated by the development of second-generation modern therapeutic intervention as far as disease (cancer) treatments is concerned (Cross & Burmester).

Cross, Deanna., & James, Burmester. “ Gene Therapy for Cancer Treatment: Past, Present and Future .” Clinical Medicine and Research . 2006. Web.

Gardlik, R., Behuliak, M., Palffy, R., Celec, P., & Li, C. “Gene therapy for cancer: bacteria- mediated anti-angiogenesis therapy.” Clinigene Current Gene Therapy Weekly (2011): 7.

Grammatikakis, I., Zervoudis, S., & Kassanos, D. “Synopsis of new antiangiogenetic factors, mutation compensation agents, and monoclonal antibodies in target therapies of breast cancer.” Clinigene Current Gene Therapy Weekly (2011): 7.

Li, Y., Li, M., Yao, G., Geng, N., Xie, Y., Feng, Y., Zhang, P., et al. “Telomerase inhibition strategies by siRNAs against either hTR or hTERT in oral squamous cell carcinoma.” Clinigene Current Gene Therapy Weekly (2011): 4.

Rothchild, Allisa., Laura, Martin., & Lauren, Lubrano. “Gene Therapy and the Gametes.” Somatic Gene Therapy . 2010. Web.

Walesgenepark.co.uk . “What is Germline Gene Therapy.” Wales Gene Park . 2011. Web.

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1. IvyPanda . "Gene Therapy: Risks and Benefits." April 18, 2021. https://ivypanda.com/essays/gene-therapy-risks-and-benefits/.

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IvyPanda . "Gene Therapy: Risks and Benefits." April 18, 2021. https://ivypanda.com/essays/gene-therapy-risks-and-benefits/.

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• Published: 08 November 2023

Successes and challenges in clinical gene therapy

• Donald B. Kohn   ORCID: orcid.org/0000-0003-1840-6087 1 , 2 , 3 ,
• Yvonne Y. Chen 1 , 4 , 5 &
• Melissa J. Spencer 3 , 6  

Gene Therapy volume  30 ,  pages 738–746 ( 2023 ) Cite this article

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Despite the ups and downs in the field over three decades, the science of gene therapy has continued to advance and provide enduring treatments for increasing number of diseases. There are active clinical trials approaching a variety of inherited and acquired disorders of different organ systems. Approaches include ex vivo modification of hematologic stem cells (HSC), T lymphocytes and other immune cells, as well as in vivo delivery of genes or gene editing reagents to the relevant target cells by either local or systemic administration. In this article, we highlight success and ongoing challenges in three areas of high activity in gene therapy: inherited blood cell diseases by targeting hematopoietic stem cells, malignant disorders using immune effector cells genetically modified with chimeric antigen receptors, and ophthalmologic, neurologic, and coagulation disorders using in vivo administration of adeno-associated virus (AAV) vectors. In recent years, there have been true cures for many of these diseases, with sustained clinical benefit that exceed those from other medical approaches. Each of these treatments faces ongoing challenges, namely their high one-time costs and the complexity of manufacturing the therapeutic agents, which are biological viruses and cell products, at pharmacologic standards of quality and consistency. New models of reimbursement are needed to make these innovative treatments widely available to patients in need.

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Successes and challenges in gene therapy—inherited blood cell disorders, hematopoietic stem cell transplantation (hsct) for inherited blood cell diseases.

Inherited blood cell diseases were the first group of disorders approached and successfully treated with gene therapy. These monogenic diseases affect the production of blood cells or their function and include 1) hemoglobinopathies that affect red blood cells (sickle cell disease, thalassemia); 2) inborn errors of immunity (IEI) affecting neutrophils, macrophages or lymphocytes; 3) lysosomal storage diseases and some leukodystrophies affecting tissue resident macrophages and brain microglial cells, and 4) conditions that lead to impaired HSC function and genome stability (Fanconi Anemia).

These inherited blood cell diseases can be cured by transplanting normal hematopoietic stem cells (HSC) from a suitably matched healthy (allogeneic) donor that can engraft and produce normal blood cells. The donor HSC are infused into the bloodstream of the patient and will home to the bone marrow space, where they take up life-long residence and produce the needed blood cells that were deficient. Performing HSC transplantation for blood cell diseases requires the availability of a suitably matched donor to minimize risks of unwanted immunological reactions between the immune cells of the donor and the recipient, such as graft rejection or graft versus host disease. The outcomes of HSC transplants have progressively improved over the last several decades, due to improved methods for tissue typing, better methods for conditioning the recipient with chemotherapy to “make space” for the engraftment of the donor HSC in the bone marrow, and improved supportive measures, such as antibiotics, antifungal and antiviral drugs, and nutritional support. However, lack of suitable matched donors and the immunological complications limit outcomes for allogeneic HSC transplantation.

Hematopoietic stem cell gene therapy (HSCGT)

Hematopoietic stem cell gene therapy ( HSCGT ) for inherited blood disorders uses the patient’s own (autologous) HSC that are gene corrected either by adding a normal copy of the inherited defective gene with an integrating vector or, more recently, editing the defective gene to restore its function. Because HSC can be removed from the body by bone marrow harvest or blood stem cell collection, gene modified ex vivo, and then returned to the patient by intravenous infusion, relatively high levels of engraftment of gene-corrected HSC can be achieved (25->90%). HSCGT has shown clinical efficacy for a growing list of disorders (Table  1 ) [ 1 , 2 ] and there are many additional related disorders with gene-specific therapies for them under early clinical or pre-clinical development. HSCGT for X-linked adrenoleukodystrophy (Skysona™) and β-thalassemia (Zynteglo™), recently received U.S. FDA marketing approval.

Severe combined immune deficiency (SCID) was the first clinical success with gene therapy. SCID is the most severe human IEI, with absent T and B lymphocyte function making the infant susceptible to life-threatening infections with high mortality in the absence of treatment. It is the first genetic blood cell disease to be curatively treated with allogeneic HSC transplantation, with bone marrow transplants from matched siblings being highly effective at restoring immunity [ 3 , 4 ]. But the majority of SCID patients do not have a matched sibling donor and are treated with transplants from alternative donors including haplo-identical parents, or well-matched unrelated donors. Survival with sustained immunity after these types of transplants has been lower than with sibling donors, but outcomes are continually improving [ 4 , 5 ]. Nevertheless, autologous gene therapy may provide an effective and safe treatment.

Initial successes to restore immunity were achieved using murine gamma-retrovirus vectors to transfer the genes for XSCID ( IL2RG ) and ADA SCID ( ADA ) into patient’s bone marrow HSC, with clinically beneficial immune reconstitution and good health [ 6 , 7 ]. However, two or more years after gene therapy, 6 of 20 XSCID patients developed a serious complication of leukemia induced by the vector [ 8 , 9 ], and once recently for ADA SCID [ 1 ].

The field shifted to the use of lentiviral vectors for their lower risks for genotoxicity compared to gamma-retroviral vectors, as well as more effectiveness for transducing human HSC. In fact, the recent clinical results with gene therapy for XSCID, ADA SCID as well as Artemis SCID ( DCLRE1C ) using lentiviral vectors have been excellent [ 10 , 11 , 12 , 13 ]. Across multiple studies in France, the U.K. and the U.S. there are consistently very high frequencies of survival with successful immune reconstitution. There is also lower transplant acuity with gene therapy compared to alternative donor transplants because reduced intensity conditioning is used, without immune suppression. Other IEIs have been successfully treated with HSCGT using lentiviral vectors, including Wiskott-Aldrich syndrome, chronic granulomatous disease, and leukocyte adhesion deficiency I [ 14 , 15 , 16 , 17 ].

Excellent clinical results have also been achieved for several metabolic disorders (lysosomal storage and leukodystrophies) in which monocyte-derived cells—macrophages, microglia—are involved in disease pathogenesis [ 18 , 19 , 20 ]. In several of these disorders, (e.g., metachromatic leukodystrophy, mucopolysaccharidosis I) the transgene leads to over-expression of the gene product which can be released from the transduced blood cells and cross-correct other somatic cells. This over-expression and high-level cross-correction do not occur using allogeneic healthy donors, and thus HSCGT may produce superior results for these diseases, as has been indicated for metachromatic leukodystrophy [ 20 ].

The hemoglobinopathies, sickle cell disease and β- and α-thalassemia, are important disease targets for gene therapy as these disorders are more common than the IEI and metabolic disorders. For β-thalassemia, lentiviral vectors have been developed expressing β-globin genes that supplement the deficient endogenous β-globin production. The published trials of one lentiviral vector for β-thalassemia showed high rates of improvement in red blood cell production, to allow transfusion therapy to be stopped for most treated patients [ 21 , 22 ]. This is the treatment for β-thalassemia HSGCT (Zynteglo™) that was recently approved by the U.S. FDA, mentioned above.

Most gene therapy approaches for sickle cell disease are based on the clinical observation that expression of increased amounts of fetal hemoglobin moderate the severity of sickle cell disease, attributed to the ability of fetal hemoglobin to slow the rate of aggregation of deoxyhemoglobin S [ 23 , 24 ]. The γ-globin chain of fetal hemoglobin has a specific amino acid (Q87) that is responsible for interference with HbS aggregation. Lentiviral vectors expressing “anti-sickling genes” (γ-globin transgene or β-globin substituted with the Q87 amino acid from γ-globin) that impede aggregation of sickle hemoglobin have been shown to have clinical benefits, with significant reduction in acute complications of sickle cell disease [ 25 , 26 ]. Other approaches used a short hairpin RNA to reduce the mRNA for the repressor of γ-globin expression, BCL11a , inducing high levels of fetal globin production and greatly reducing acute sickle complications [ 27 ]. CRISPR/Cas9 is being used to disrupt the erythroid enhancer element for the BCL11a gene to induce increased γ-globin production and to facilitate correction of the sickle cell-causing mutation in the HBB S gene by homology-directed repair [ 28 , 29 , 30 ]. Base-editing trials advancing to the clinic will be used to either eliminate the BCL11a-binding sites at the γ-globin genes and thereby induce fetal globin; or to convert the codon containing the sickle cell-causing mutation to one encoding an amino acid that does not cause sickling [ 31 , 32 ].

Opportunities

There are many more inherited blood cell diseases for which lentiviral vector gene therapies are being developed, including additional IEI, α-thalassemia, storage and metabolic disorders. Gene editing technologies are advancing at break-neck speed, with nuclease-mediated editing (zing finger nucleases, TALENS, CRISPR/Cas9) being followed by base editing, prime editing and more to come. The ability to make precise edits in endogenous genes should confer more physiological expression of genes needed for safe and effective HSCGT.

Safety risks

In terms of safety issues, a hypothetical concern about lentiviral vectors recombining with the lentiviral genes used for packaging to produce a replication-competent lentivirus capable of spreading infection has never been reported [ 33 ].

A major safety concern for HSCGT is the risks of genotoxicity from the integrating vector. In fact, early trials using gamma-retroviral vectors for XSCID, CGD, and WAS had serious complications from the vector causing leukemia in some of the patients [ 1 , 2 ]. Lentiviral vectors have been markedly safer across multiple clinical trials for more than a dozen disorders. In general, integration site analyses do not show preferential integration near oncogenes, nor clinically significant clonal expansion. The only clinically significant genotoxicities with lentiviral vectors have occurred with vectors that contained the long terminal repeat enhancer elements from gamma-retroviruses, which are the element responsible for their genotoxicity; or with inclusion of an insulator element that inadvertently acts as a splice acceptor-polyA signal and can cause premature truncation of cellular transcripts when the vector integrates into introns [ 34 , 35 ]. Vectors of many other designs, using promoters of cellular housekeeping genes with low enhancer activity (e.g. human phosphoglycerate kinase, or elongation factor-1α) or with lineage-restricted expression (e.g. beta-globin, chimeric myeloid) have not shown genotoxicity, but yield a polyclonal vector distribution without clonal expansions [ 1 , 2 ].

It is postulated that use of site-specific gene editing may have significantly reduced risks of genotoxicity compared to randomly integrated lentiviral vectors. However, gene editing methods also have inherent genotoxicity risks, including disruptive insertions and deletions of various sizes at editing sites, loss of chromosomal material distal to a nuclease cleavage site, bystander edits in the region targeted by base editors, and off-target editing. The risks for any specific editing strategy need to be considered and assessed as part of clinical translation of gene editing for a clinical cell product.

The manufacture of gene-modified autologous HSC drug products is complex. A patient-specific stem cell collection is needed for each autologous product. These procedures have moderate clinical complexity, entailing 5–7 days of receiving G-CSF for mobilization, placement of a suitable central venous pheresis catheter, and 1–3 leukapheresis sessions.

CD34 + cell selection and either lentiviral vector transduction or gene editing with reagents introduced by electroporation are relatively standardized, but entail many hours of cell processing in the clean room facility. There is a relatively low rate of lot failure, with criteria for purity, potency, identity and safety met in most cases. And, administration of the gene-modified stem cells in the context of a clinical stem cell transplantation with moderate to intense conditioning chemotherapy requires high acuity in-patient medical care for several weeks. However, the clinical standard of care approach for these disorders is an allogeneic HSCT which has similar (or greater) clinical complexity.

Conditioning

Conditioning chemotherapy is routinely used as pre-transplant in nearly all blood-cell diseases to attain sufficient engraftment of the gene-modified HSCs, with Fanconi anemia being an exception that can achieve therapeutic levels of stem-cell engraftment without conditioning due to the stem-cell defects inherent in the disease [ 36 ]. The conditioning imposes risks of organ toxicity, infections, pancytopenia requiring transfusions and often antibiotics, as well as discomforts including mucositis, nausea, vomiting, anorexia and alopecia. While conditioning chemotherapy also carries risks from potential mutagenic effects of the alkylating agents, at least one report of a cohort of ADA SCID patients who received reduced-intensity conditioning with busulfan did not display any mutations in the blood cells typical of clonal hematopoiesis [ 37 ]. Nevertheless, there is a great deal of effort to replace chemotherapy with less toxic conditioning agents, such as monoclonal antibodies to stem cell markers (e.g., CD117, CD45), either unconjugated or as antibody-drug conjugates [ 38 , 39 , 40 ]. Effective conditioning without chemotherapy should significantly improve the safety profile of these autologous transplants.

Variability

There is significant variability in the levels of engraftment of gene-corrected cells across members in different study cohorts in reported gene therapy trials [ 14 , 15 , 19 , 26 ]. We have shown that, in an ADA trial, the level of engraftment of gene-modified stem cells (based on vector copy number in granulocytes) was a function of the CD34 + cell dose, the percentage of the CD34 + cells that were transduced, and the intensity of the conditioning, based on the area-under-the-curve for busulfan exposure [ 41 ]. Thus it is important to optimize each component of the gene therapy procedure for optimal results.

HSCGT is delivered in the context of autologous HSC transplant, and the costs for the clinical component are relatively standard, including screening, clinical labs, central venous line placement, administration of conditioning chemotherapy, the post-transplant clinical care including the costs for in-patient stay on ICU-like transplant units plus costs for antibiotics, parental nutrition if needed, lab and infectious disease testing, radiology studies, etc. The unique components of cost are those for manufacturing the cell product, including the vector lot, the cell processing costs (materials for CD34 selection, cell culture, testing, GMP facility, regulatory oversight). In the academic setting many of these costs are much lower than in the commercial setting where the expectations for the quality of the documentation, facility building costs, maintenance and oversight, and a much higher level of staffing raise the costs.

Commercialization

The major challenges to developing and employing these gene therapies are not technical but financial. The costs are high for reagents like clinical-grade lentiviral vectors or gene editing reagents, as well as for the cell processing materials and Good Manufacturing Practices (GMP) facility and personnel costs, in addition to the drug research and development costs. It is anticipated that costs per patient dose may be reduced as the methods for vector production and cell processing are improved. The improved safety and reduction in clinical costs and improved outcomes using these autologous gene-corrected HSC products need to exceed those from the various allogeneic HSCT options that are available. Certainly, avoiding the use of potent immune suppressive drugs pre- and post-transplant and the absence of risks for graft versus host disease may provide this competitive edge to gene therapies, but this is to be determined.

Successes and challenges in gene therapy—chimeric antigen receptor T cells (CARs)

Redirecting immune cells against cancer.

Immunotherapy, or treatment that engages or redirects the immune system against diseased cells, has become a fourth pillar in cancer immunotherapy alongside chemotherapy, radiation, and surgery. In addition to small-molecule drugs that interface with immune-cell function [ 42 ] and protein biologics such as cytokines and checkpoint-blockade antibodies [ 43 , 44 ] cell-based immunotherapy has emerged as a potent new tool in the immuno-oncology arsenal. Here, we provide a brief overview of the successes and challenges of genetically modified cell-based therapy for cancer.

Cell-based immunotherapy can be conceptualized as the engineering of a chassis (i.e., the immune cell) to execute anti-tumor operations with the assistance of programmed software (i.e., transgenic elements that encode specified functions). The development of antigen-specific receptors that redirect engineered immune cells against tumor cells forms the foundation of cell-based cancer immunotherapy. Various immune cell types—including T cells, natural-killer (NK) cells, and macrophages—have been engineered to express tumor-targeting receptors, expanded ex vivo, and infused into cancer patients to achieve targeted tumor eradication[ 45 , 46 , 47 ]. To date, T cells serve as the most commonly used chassis in cell-based immunotherapy, and two main categories of receptors have been used to redirect T-cell specificity toward cancer: T-cell receptors ( TCRs ) and chimeric antigen receptors ( CARs ).

TCRs are naturally occurring receptors that define the antigen specificity of T cells. Pioneering work in T-cell therapy led to the discovery that tumor-infiltrating lymphocytes (TILs), which express endogenous TCRs recognizing tumor-specific or tumor-associated antigens, can be isolated from cancer patients, expanded ex vivo, and re-infused into the same patient to augment the antitumor response [ 48 , 49 ]. Extending beyond TIL isolation and expansion, one could identify tumor-reactive clones among TILs, isolate the tumor-reactive TCR sequence, and introduce a transgenic copy of the TCR gene into non–tumor-specific T cells to artificially confer tumor-recognition capability [ 50 , 51 ]. Furthermore, a suite of technologies has been developed to identify and isolate tumor-reactive TCRs via high-throughput in vitro library screening. For example, multiplexed single-cell transcriptomics and TCR sequencing yielded the identification of TCR clones that recognize a “public” neoantigen derived from PIK3CA , with clinical applicability to patients with diverse malignancies ranging from uterine serous carcinoma to colon adenocarcinoma to anaplastic thyroid cancer [ 52 ]. This approach begins with a known target antigen, and search for cognate TCRs. The reverse process—i.e., starting with a tumor-reactive TCR in search of a cognate ligand—can also be accomplished through the use of peptide-major histocompatibility complex (MHC) libraries presented by target cells [ 53 ].

Unlike TCRs, CARs are synthetic proteins comprising heterologous parts, and they can recognize antigens in an MHC-independent manner [ 54 ]. Importantly, CARs have been shown to function not only in T cells, but also in NK cells [ 55 ], macrophages [ 56 ], and neutrophils [ 57 ]. The antigen-specificity of CAR molecules is dictated by its extracellular ligand-binding domain, which is most commonly a single-chain variable fragment derived from a monoclonal antibody that binds the antigen of interest. The ligand-binding domain is connected via an extracellular spacer to a transmembrane domain, followed by cytoplasmic signaling domains such as the CD3ζ chain and co-stimulatory domains such as CD28 and 4-1BB. In August 2017, Kymriah TM (tisa-cel) became the first genetically modified cell therapy for cancer to receive FDA approval. Tisa-cel is an autologous CAR-T cell product targeting CD19, a pan–B-cell marker present on all mature B cells as well as the majority of malignant B cells. In its registration trial for the treatment of pediatric and young adult patients with relapsed or refractory B-cell acute lymphocytic leukemia (B-ALL), Kymriah TM achieved 82% (65/79) overall remission rate and a 66% probability of relapse-free survival at 18 months [ 58 ]. Since 2017, several additional autologous CAR-T cell therapies targeting CD19 or BCMA have been approved for the treatment of B-cell leukemia, lymphoma, and multiple myeloma (Table  2 ) [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ]. As the technology matures, CD19 CAR-T cell therapy has advanced from third-line to second-line treatment for non-Hodgkin lymphoma, and clinical trials are already underway to evaluate CD19 CAR-T cell therapy as first-line treatment for high-risk large B-cell lymphoma [ 69 ]. In addition, CD19 CAR-NK cells have also demonstrated clinical promise [ 70 ], elevating NK cells as another potent effector cell type for immunotherapy.

The success of CD19- and BCMA-targeted therapies highlight not only the promise but also the challenges associated with cell-based immunotherapy. Specifically, despite clinical trials evaluating CAR-T cells targeting dozens of antigens, only CD19 and BCMA CAR-T cells have received FDA approval to date, and no cell-based therapy has yet demonstrated comparable efficacy against solid tumors. To expand the applicability of cell-based therapy to a broad range of malignancies, engineering efforts now focus on not only optimizing the CAR protein, but also fine-tuning immune-cell biology to maximize anti-tumor efficacy.

Genetic modifications to enhance T-cell potency

To achieve safe, potent, and durable therapeutic benefit, cell-based therapies must meet a number of performance criteria: (i) precise targeting of tumor cells and simultaneous avoidance of essential healthy tissue, (ii) complete coverage of the tumor population based on antigen recognition, and (iii) robust expansion and functional persistence of the effector cell population to eradicate existing tumor burden and provide long-term surveillance against tumor relapse. The first two objectives are most typically achieved through careful antigen selection and receptor engineering, a topic that has been extensively covered in several reviews[ 71 , 72 ]. Here, we discuss strategies aimed at promoting the expansion and functional persistence of engineered cells post-infusion.

Different immune cell types are driven by fundamentally divergent biology that necessitates different strategies to optimize for therapeutic applications. T cells, as the dominant chassis to date, has been the focus of most engineering efforts in this area. T cells are a uniquely interesting substrate for engineering due to their natural diversity in phenotype and function. For example, effector T cells can execute potent cytotoxicity and explosive growth, but are relatively short-lived. In contrast, memory T cells are more tempered in effector functions, but can provide long-term persistence and in vivo surveillance. Importantly, despite efforts to neatly categorize T cells into distinct subtypes, advancements in epigenomic, transcriptomic, and proteomic analyses continue to reveal subtle gradations in T-cell biology and function [ 73 ]. For example, exhausted T cells, long described as a dysfunctional subset, are now understood to play a critical role in the effectiveness of immune checkpoint blockade therapy [ 74 ]. This increasingly complex picture of T-cell phenotype presents both challenges and opportunities for engineering, as it may be possible to genetically tune T-cell behavior to obtain an optimal combination of functions conducive to cancer therapy.

Early efforts in optimizing CAR-T cells’ anti-tumor efficacy focused on introducing co-stimulatory signaling into the CAR molecule, thus promoting T-cell proliferation and the intensity of effector outputs such as cytokine production and cytotoxicity upon CAR signaling [ 75 ]. With accumulating clinical experience, a number of genetic targets were identified as potential targets for ablation to improve T-cell function. For example, the success of anti–PD-1 checkpoint blockade led to the first US-based clinical trial involving CRISPR-edited cell therapy, in which T cells expressing an anti–NY-ESO-1 TCR were also edited by CRISPR/Cas9 to knock out PD-1 [ 76 ]. As another example, a patient with chronic lymphocytic leukemia responded to anti-CD19 CAR-T cell therapy only upon receiving a second dose of CAR-T cells, and the anti-tumor efficacy was attributed in large part to clonal expansion of a particular CAR-T cell that lacked any functional copy of TET2 (the patient had a congenital mutation in one copy of TET2, and the CAR transgene was randomly inserted into the other copy, thereby generating a double-knockout) [ 77 ]. This subsequently led to the intentional knockout of TET2 as a means to promote in vivo T-cell expansion and persistence [ 78 ]. More recently, the advent of CRISPR-based genetic libraries has enabled large-scale screening efforts to identify target genes whose knock-in or knock-out can enhance T-cell expansion, persistence, and anti-tumor efficacy. For example, the SWI/SNF family complex member ARID1A and the RAS GTPase-activating protein RASA2 have been identified through library screens and subsequently individually validated as knockout candidates to minimize T-cell exhaustion and improve T-cell function [ 13 , 79 ]. Finally, numerous strategies are now under evaluation to “armor” tumor-targeting immune cells with chemokines, cytokines, and genetic circuitry designed to enhance anti-tumor efficacy, particularly against solid tumors that are otherwise protected by highly immunosuppressive tumor microenvironments [ 80 , 81 ].

Manufacturing challenges in cell-based immunotherapy

As additional gene targets are identified, technical challenges surrounding cell manipulation are rising to the forefront of the clinical translation process. To date, the vast majority of CAR-expressing immune cells, regardless of cell type, undergo lenti- or retroviral transduction to introduce the CAR-encoding transgene. However, the payload limitations of viral vectors quickly become a bottleneck when additional genetic elements need to be knocked in or knocked out of the cell product. Furthermore, the manufacturing of clinical-grade viral vectors is a major roadblock due to the high cost and limited availability of manufacturing slots. To overcome this chokepoint in therapy development, active research focuses on developing non-viral methods of gene delivery, including transposon-mediated gene integration [ 82 ], CRISPR-mediated homologous recombination [ 83 ], nanoparticle-mediated in situ gene transfer [ 84 ], among others.

The personalized nature of autologous therapy presents a significant manufacturing challenge, as a new product must be generated for each individual patient. Furthermore, most patients have already experienced multiple rounds of prior therapy with the potential to damage their immune cells, rendering the cell manufacturing process even more precarious. Allogeneic therapy using donor-derived cells present an attractive alternative to on-demand generation of patient-specific cell products, as the starting cell population from healthy donors could have substantially higher fitness levels compared to patient cells. In principle, the ability to pre-manufacture the cell product would also enable more extensive quality-control testing, potentially more complex genetic engineering, and the generation of sufficient doses for multiple patients from a single manufacturing campaign. To date, experimental allogeneic cell therapies have included αβ T cells with endogenous TCRs knocked out to minimize graft-versus-host, γδ T cells, NK cells, and invariant natural killer T (iNKT) cells; however, emerging data on allogeneic cell therapy indicate that durability of response remains a key challenge [ 85 , 86 ].

Successes and challenges in gene therapy—adeno-associated virus (AAV) therapies

Viral-based gene delivery systems rely on the natural ability of viruses to infect cells and deliver their genetic cargo. Recombinant Adeno-Associated Viral vectors ( AAVs ) are the preferred delivery vehicle for a number of gene therapies, due to their small size, high efficiency, low immunogenicity, and tunable tissue tropism. Wild type (WT) AAV is a non-enveloped parvovirus comprised of a single strand of DNA with Rep and Cap genes. Rep encodes four proteins that control viral replication, packaging, and genomic integration, and Cap encodes three subunits called VP1, VP2, and VP3, which make up the capsid coat in ratios of 1:1:10. The WT AAV genome is flanked by inverted terminal repeats (ITRs), which are critical for the replication and packaging of the DNA cargo within the capsid coat. AAV is so-named because it was initially identified as a contaminant in adenoviral preparations, and is not associated with any known disease. Typically, AAV-based gene therapy involves delivering a functional copy of a gene to replace a non-functional version; however, any nucleic acid can theoretically be delivered by AAV. AAV’s flexibility for different gene therapy applications is being increasingly recognized and adapted to deliver other therapeutic molecules, such as regulatory RNAs and gene editing platforms. Currently, the majority of gene-therapy products and products in late-stage clinical development use a gene replacement approach (Table  3 ). There are three approved AAV gene therapies in the United States (Luxturna TM , Zolgensma TM , and Hemgenix TM ), and one with conditional approval in Europe (Rocktavian TM ), but many more are being developed and trials are ongoing. While AAV-based approaches have been curative for some diseases, safety, efficacy, and manufacturing challenges remain.

Basics of vector design for AAV based gene therapy

Design of the aav cargo.

In order to use an AAV for gene therapy applications, the AAV genome ( Rep and Cap gene) is removed, and replaced by a therapeutic expression cassette. This cassette consists of a regulatory element (promoter), the cDNA encoding the functional gene, and a poly A signal. The cassette is flanked by the only two remaining viral genome elements, the two 140 bp ITRs. The total size of the expression cassette including the ITRs should be ~4.8 kb, although larger inserts at ~5 kb have been successfully packaged at a cost of reduced efficiency. By using a tissue-specific promoter, transgene expression is limited to the target tissue(s), preventing expression in antigen-presenting cells, which can trigger transgene-specific immune responses. The therapeutic transgene can be codon optimized to improve its translation; however, this modification often increases the number of unmethylated CpGs, which can contribute to enhanced immune responses to the transgene [ 87 , 88 ], so codon optimization should be carried out without increasing the CpG content.

Design of the AAV capsid

The AAV capsid determines the tissue specificity or “tropism” for each AAV serotype. Hundreds of AAV serotypes of human or primate origin have now been described. While the mechanisms are not completely elucidated, most AAV serotypes use a co-receptor (often a membrane protein) and receptor (a glycan) to gain entry to cells. Upon receptor and co-receptor binding, AAV is endocytosed, escapes the endosome, travels to the nucleus and is uncoated. In the nucleus, the single-stranded DNA is converted to double-stranded DNA, circularized, and subsequently remains as an episome. The capsid proteins are degraded in the proteasome and presented on major histocompatibility complexes. New AAV serotypes are being identified through capsid engineering to create serotypes with enhanced specificity to tissues of interest [ 89 ]. AAV2 has the widest tissue tropism of all known serotypes, likely because it is able to utilize numerous receptors and co-receptors. The choice of AAV capsid for gene therapy applications should target disease-relevant tissues with as much specificity as possible.

AAV packaging

For gene therapy applications, AAV packaging is accomplished in human (HEK293) or insect (sf9) cells, which can be adherent or grown in suspension. Use of these two cell types results in differences in the types of post-translational modifications that are present after packaging, as well as the integrity of the packaged DNA [ 90 , 91 ]. Because the genes involved in replication and packaging are removed from the expression cassette, the Rep and Cap genes need to be supplied in trans along with an adenoviral helper plasmid to accomplish AAV packaging. Thus, three plasmids are triple transfected to cells in order to package AAV. Following packaging, a variety of purification methods can be used, including cesium chloride centrifugation [ 92 ], ion exchange chromatography [ 93 ], and affinity purification [ 94 ]. It is common for contract development and manufacturing organizations (CDMOs) to handle this task for GMP-grade vector, although many companies are developing their own manufacturing capabilities.

Luxturna TM for Leber congenital amaurosis (LCA): In 2017, the first AAV-based gene therapy was FDA-approved for the treatment of a form of hereditary blindness called LCA. LCA is comprised of 23 different genetically defined retinal disorders that lead to vision loss [ 95 ]. The drug Luxturna is now approved for LCA patients harboring mutations in both alleles of the RPE65 gene [ 96 ]. Mutations affect the production or function of RPE65, which is expressed in retinal epithelial cells. The RPE65 gene encodes a retinoid isomerohydrolase that is needed to produce a chromophore for phototransduction, called 11-cis retinal [ 97 ]. Photoreceptors lacking RPE65 will degenerate and lead to vision loss. Individuals with homozygous mutations experience progressive vision loss during the first year of life. Luxturna uses AAV2 to carry the human RPE65 gene under the control of the beta actin promoter, leading to dramatic restoration of vision to those who receive this therapy.

Zolgensma TM for Spinal Muscular Atrophy type I: The second AAV-based gene therapy approved in the United States is Zolgensma TM , which received FDA approval in May 2019 for children under the age of 2 with infantile-onset spinal muscular atrophy (SMA) type I (AKA Werdnig-Hoffman disease). Zolgensma TM is indicated for babies with homozygous loss of function mutations in the SMN1 gene. SMN is a protein that is necessary for development of alpha motor neurons. In the absence of SMN protein, the spinal cord and brainstem degenerate, leading to weakness in the limbs, trunk, swallowing and breathing muscles. Children born with SMA type I are not able to sit unassisted, have difficulty breathing and swallowing, and usually die within the first year of life. Zolgensma TM uses AAV9 to deliver a copy of the SMN1 gene, driven by a CMV promoter. Because SMN expression is needed in both neurons and muscle cells, the therapy utilizes a promoter with broad tissue expression. This therapy appears to be most efficacious if administered prior to 6 months of age because post-natal delivery of SMN1 stabilizes, but does not reverse the disease process. However, treatment with Zolgensma TM has been life-changing for children born with SMA type I, with many able to breathe, eat and even walk on their own. Reports five years post-dosing suggest that the durability of Zolgensma TM is high. This therapy was the first systemically delivered AAV-based therapy and as such, has paved the way for many more that require intravenous administration and systemic delivery.

Roctavian TM for Hemophilia A and Hemgenix TM for Hemophilia B: Hemophilia is a rare inherited blood clotting disorder caused by deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B). Patients experience episodes of excessive bleeding affecting soft tissues and joints. Gene therapy programs to restore these factors are being actively pursued in both disease categories; however the first commercially successful program was achieved in hemophilia A [ 98 ]. Roctavian TM has EMA conditional approval for treatment of hemophilia A, which uses AAV5 to deliver a FVIII cDNA under the regulation of a liver specific promoter. The therapy restores FVIII at high doses, resulting in reduced need for externally provided FVIII (99% reduction). However, in the phase III study, loss of transgene is apparent with time, from an average of 64% of FVIII activity at 1 year down to 24% by year 4 (J.P. Morgan presentation 11 Jan 2021). The reason for the loss of transgene is not clear, but may relate to the high immunogenicity of FVIII or natural turnover of hepatocytes with time. Shortly after Rocktavian’s conditional EMA approval in 2022, for hemophilia A, Hemgenix TM received FDA approval for hemophilia B. Like Roctavian TM , Hemgenix TM . Uses the AAV5 vector to deliver FIX under the control of a liver promoter.

The immune response to AAV poses the greatest challenge for successful AAV-based therapies. Three components of AAV vectors can trigger immunity: 1) AAV-capsid, 2) unmethylated CpGs in the nucleic acid cargo and 3) the protein transgene. Immune responses prevent vector re-administration, thus limiting AAV to a single dose. Furthermore, because humans are naturally exposed to wild type AAV (wtAAV), an estimated 30–70 % of individuals have pre-existing immunity by the time they reach adulthood [ 99 , 100 ]. Therefore, successful AAV-based therapies require that AAV vectors overcome preexisting immune responses in patients. Strategies to dose in the presence of pre-existing immunity have thus far been unsuccessful, but researchers are testing whether plasmapheresis [ 101 ], capsid decoys [ 102 ], or the use of enzymes to cleave circulating IgG will have utility [ 103 ].

Safety and toxicity

Although clinical trials with AAV are still in their early stages, AAV is generally considered safe when dosed in vivo. Most AAV serotypes are sequestered in the liver and as such the most common adverse event is liver toxicity, clinically observed as an elevation of liver enzymes [ 104 , 105 , 106 , 107 ]. In hemophilia gene therapy trials, patients experienced liver toxicity that could be resolved with a short course of prednisone treatment [ 108 ]. These SAEs were attributed to CD8 T cell mediated attack against hepatocytes presenting capsid on MHC [ 106 , 109 ]. Steroid treatment could be discontinued once the viral capsid was shed [ 110 ]. Other adverse events include hepatic hepatocellular carcinoma, which was observed in a single patient who also had several co-morbidities, and this event led to a pause in a clinical trial for hemophilia B.

The most prevalent adverse events (AEs) in high-dose AAV administration are thrombotic microangiopathy (TMA) or atypical hemolytic uremic syndrome (aHUS), both of which are likely due to complement recognition of the AAV capsid. TMA has been observed in several spinal muscular atrophy and Duchenne muscular dystrophy (DMD) subjects treated with high-dose, systemically delivered AAV vectors. Based on reports from Pfizer, Solid Biosciences, and Novartis [ 111 , 112 ], there is ample evidence that this adverse event likely relies on the presence of anti-AAV antibodies [ 113 ]. TMA has led to clinical holds for two different DMD trials, but symptoms were resolved using eculizumab, a C5 complement inhibitor, for most patients, although two DMD patients died. Trials for X-linked microtubular myopathy, in which AAV8 was used to deliver the MTM1 gene, have resulted in patient deaths from liver failure, which may be a component of the disease. Taken together, low-dose AAV treatment is supported by a strong safety profile, especially in the presence of steroids, but higher AAV doses appear to trigger TMA or aHUS in a subset of patients with pre-existing antibodies or rapid antibody responses. Dosing protocols are increasingly adding immunosuppression to avoid these AEs. It is important to note that the methods for vector production and titration are not standardized and so dosages, empty to full capsid ratios and impurities (such as endotoxins) could differ across trials and may be a contributing factor to some of these toxicities.

While AAV-based approaches have been considered to be “one and done”, all available evidence suggests that re-dosing of the therapeutic will be needed to achieve a long-lasting therapy. The durability of the transgene can be compromised by the lifespan of the target cell or the immune response to the transgene, but for each target tissue and vector, these risks will need to be established independently. Since an adaptive immune response develops after initial dosing, new doses of vector will be neutralized before reaching its target tissue. Changing AAV capsids does not seem to be a valid solution for avoiding immunity, because of the high capacity for cross-reactivity. Therefore AAV-based approaches are limited to a single dose unless additional measures are taken to reduce antibodies and T cells from prior exposure. Re-dosing can be achieved if the initial dose is provided in the presence of strong immunosuppression, but these studies are still in their early stages [ 114 , 115 ]. Transgene immunity can be a major challenge for treating patients with null mutations due to lack of tolerization of the transgene product, which has been observed in hemophilia A [ 116 , 117 ] and DMD (Bonneman, American Society for Gene and Cell Therapy, 2022 Annual meeting). Several patients developed anti-transgene responses manifesting as myositis in three different DMD gene therapy trials, and it has been suggested that the target is the transgene. This observation led several companies to change their inclusion criteria, only including patients whose endogenous gene contains the same elements that are contained within the transgene. Therefore, while initial results have been extremely promising, the issue of durability will likely need to be addressed.

Manufacturing, scale up, and costs

The production, scale-up, and costs of AAV-based therapies are major obstacles to their widespread success. This issue is particularly problematic for gene therapy applications requiring high vector doses, such as those targeting skeletal muscle [ 118 ]. There are a number of steps involved in creating the final biological product, including the source and type of cells for packaging, the manufacturing method for plasmids used for transfection, the choice of helper plasmid, the stability and purity of the final product, and determining titers and empty-to-full ratios, among others [ 119 ]. The manufacturing process and how it impacts these characteristics are largely unknown; however with enhanced transparency, the impact of manufacturing on clinical efficacy will be made clearer. Manufacturing capabilities are expanding in the US and in Europe, and it is hoped that AAV vector production will be able to meet demand as trials and commercialization progress. Currently, costs are astronomical, but as process development becomes more streamlined, it is anticipated that costs should begin to decline.

Data sharing statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

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Acknowledgements

DBK is supported by the California Institute for Regenerative Medicine and is the recipient of endowment funding from the UCLA Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research. YYC is supported by the Mark Foundation for Cancer Research, Cancer Research Institute, Parker Institute for Cancer Immunotherapy, and Jean and Stephen Kaplan. MJS is supported by the National Institutes of Health (RO1NS117912, P50AR052646), the Department of Defense, California Institute for Regenerative Medicine and the Coalition to Cure Calpain 3.

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Donald B. Kohn & Melissa J. Spencer

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Yvonne Y. Chen

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Kohn, D.B., Chen, Y.Y. & Spencer, M.J. Successes and challenges in clinical gene therapy. Gene Ther 30 , 738–746 (2023). https://doi.org/10.1038/s41434-023-00390-5

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DOI : https://doi.org/10.1038/s41434-023-00390-5

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CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future

Fathema uddin.

1 Department of Medicine, Thoracic Oncology Service, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Charles M. Rudin

2 Weill Cornell Medicine, Cornell University, New York, NY, United States

Triparna Sen

A series of recent discoveries harnessing the adaptive immune system of prokaryotes to perform targeted genome editing is having a transformative influence across the biological sciences. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy. Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity. While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy. Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation. This review focuses on the evolution of gene therapy and the role of CRISPR in shifting the gene therapy paradigm. We review the emerging data of recent gene therapy trials and consider the best strategy to move forward with this powerful but still relatively new technology.

Introduction

Gene therapy as a strategy to provide therapeutic benefit includes modifying genes via disruption, correction, or replacement ( 1 ). Gene therapy has witnessed both early successes and tragic failures in a clinical setting. The discovery and development of the CRISPR/Cas9 system has provided a second opportunity for gene therapy to recover from its stigma and prove to be valuable therapeutic strategy. The recent advent of CRISPR technology in clinical trials has paved way for the new era of CRISPR gene therapy to emerge. However, there are several technical and ethical considerations that need addressing when considering its use for patient care. This review aims to (1) provide a brief history of gene therapy prior to CRISPR and discuss its ethical dilemmas, (2) describe the mechanisms by which CRISPR/Cas9 induces gene edits, (3) discuss the current limitations and advancements made for CRISPR technology for therapeutic translation, and (4) highlight a few recent clinical trials utilizing CRISPR gene therapy while opening a discussion for the ethical barriers that these and future trials may hinge upon.

Gene Therapy Prior to Crispr—History, Hurdles, and its Future

Origins of gene therapy.

The introduction of gene therapy into the clinic provided hope for thousands of patients with genetic diseases and limited treatment options. Initially, gene therapy utilized viral vector delivery of therapeutic transgenes for cancer treatment ( 2 ) or monogenic disease ( 3 ). One of these pioneering clinical trials involved ex vivo retroviral delivery of a selective neomycin-resistance marker to tumor infiltrating leukocytes (TILs) extracted from advanced melanoma patients ( 4 ). Although the neomycin tagging of TILs did not have a direct therapeutic intent and was used for tracking purposes, this study was the first to provide evidence for both the feasibility and safety of viral-mediated gene therapy. Soon after, the first clinical trial that used gene therapy for therapeutic intent was approved in 1990 for the monogenic disease adenosine deaminase-severe combined immunodeficiency (ADA-SCID). Two young girls with ADA-SCID were treated with retroviruses for ex vivo delivery of a wildtype adenosine deaminase gene to autologous T-lymphocytes, which were then infused back into the patients ( 5 , 6 ). While one patient showed moderate improvement, the other did not ( 5 , 6 ) Although initial results were suboptimal, the early evidence of feasibility prompted multiple subsequent gene therapy trials using viral-mediated gene edition. However, this was followed by some major setbacks.

Tragic Setbacks for Gene Therapy

Jesse Gelsinger, an 18-year-old with a mild form of the genetic disease ornithine transcarbamylase (OTC) deficiency, participated in a clinical trial which delivered a non-mutated OTC gene to the liver through a hepatic artery injection of the recombinant adenoviral vector housing the therapeutic gene. Unfortunately, Jesse passed away 4 days after treatment ( 7 ). The adenovirus vector triggered a much stronger immune response in Jesse than it had in other patients, causing a chain of multiple organ failures that ultimately led to his death ( 8 ). At the time of the trial, adenoviral vectors were considered reasonably safe. In preclinical development, however, two of the rhesus monkeys treated with the therapy developed a similar pattern of fatal hepatocellular necrosis ( 9 ). Shortly after, another gene therapy trial led to the development of leukemia in several young children induced by insertional oncogenesis from the therapy ( 10 ). These trials opened for two forms of SCID (SCID-X1 or common ɤ chain deficiency) and adenosine deaminase deficiency (ADA). The therapy used ɤ-retroviral vectors for ex vivo delivery of therapeutic transgenes to autologous CD34+ hematopoietic stem cells, which were reintroduced to the patients ( 10 ). Five patients developed secondary therapy-related leukemia, one of whom died from the disease ( 11 ). Further investigation revealed integration of the therapeutic gene into the LMO2 proto-oncogene locus, presumably resulting in the development of leukemia ( 12 ). Subsequent analyses have suggested a higher frequency of insertional mutagenesis events with ɤ-retroviral vectors relative to other vectors ( 13 ). Together, these tragic events prompted substantial post-hoc concerns regarding the nature of appropriate informed consent and the stringency of safety and eligibility parameters for gene therapy experimentation in humans ( 14 ).

Shifting the Gene Therapy Paradigm

Almost two decades after these cases, gene therapy returned in clinical trials with reengineered viruses designed with safety in mind. Current clinical approaches are being scrutinized for evidence of insertional mutagenesis and adverse immunogenic reactions ( 15 – 18 ). Non-viral vectors have been used as an alternative method for gene delivery, which have reduced immunogenicity compared to their viral counterparts and therefore greater tolerance for repeated administration. A concern is whether these methods can be optimized to provide equivalent efficiency of gene delivery to that provided by viruses ( 19 ).

While viral vectors continue to be essential for current gene therapy, the concerns and limitations of viral-mediated gene edition has broadened the diversity of gene-editing approaches being considered. Rather than introducing the therapeutic gene into a novel (and potentially problematic) locus, a more attractive strategy would be to directly correct the existing genetic aberrations in situ . This alternative would allow the pathological mutation to be repaired while averting the risk of insertional oncogenesis. The discovery and repurposing of nucleases for programmable gene editing made this possible, beginning with the development of zinc finger nucleases (ZFN) ( 20 , 21 ), followed by transcription activator-like effector nucleases (TALENs), meganucleases, and most recently, the CRISPR/Cas system ( 22 ). While the other gene-editing tools can induce genome editing at targeted sites under controlled conditions, the CRISPR/Cas system has largely supplanted these earlier advances due to its relatively low price, ease of use, and efficient and precise performance. However, this technology is often delivered with adeno-associated virus (AAV) vectors, and thus does not completely avert risks associated with viruses. Other delivery options are available to circumvent this issue, each with their own advantages and challenges (see Delivery of CRISPR Gene Therapy section). Of the CRISPR/Cas systems, CRISPR/Cas9 is the most developed and widely used tool for current genome editing.

CRISPR/Cas9 Mediated Gene Editing

Pioneering discoveries in crispr/cas9 technology.

The bacterial CRISPR locus was first described by Francisco Mojica ( 23 ) and later identified as a key element in the adaptive immune system in prokaryotes ( 24 ). The locus consists of snippets of viral or plasmid DNA that previously infected the microbe (later termed “spacers”), which were found between an array of short palindromic repeat sequences. Later, Alexander Bolotin discovered the Cas9 protein in Streptococcus thermophilus , which unlike other known Cas genes, Cas9 was a large gene that encoded for a single-effector protein with nuclease activity ( 25 ). They further noted a common sequence in the target DNA adjacent to the spacer, later known as the protospacer adjacent motif (PAM)—the sequence needed for Cas9 to recognize and bind its target DNA ( 25 ). Later studies reported that spacers were transcribed to CRISPR RNAs (crRNAs) that guide the Cas proteins to the target site of DNA ( 26 ). Following studies discovered the trans-activating CRISPR RNA (tracrRNA), which forms a duplex with crRNA that together guide Cas9 to its target DNA ( 27 ). The potential use of this system was simplified by introducing a synthetic combined crRNA and tracrRNA construct called a single-guide RNA (sgRNA) ( 28 ). This was followed by studies demonstrating successful genome editing by CRISPR/Cas9 in mammalian cells, thereby opening the possibility of implementing CRISPR/Cas9 in gene therapy ( 29 ) ( Figure 1 ).

Hallmarks of CRISPR Gene Therapy. Timeline highlighting major events of traditional gene therapy, CRISPR development, and CRISPR gene therapy. The text in red denotes gene therapy events which have raised significant ethical concerns.

Mechanistic Overview of CRISPR/Cas9-Mediated Genome Editing

CRISPR/Cas9 is a simple two-component system used for effective targeted gene editing. The first component is the single-effector Cas9 protein, which contains the endonuclease domains RuvC and HNH. RuvC cleaves the DNA strand non-complementary to the spacer sequence and HNH cleaves the complementary strand. Together, these domains generate double-stranded breaks (DSBs) in the target DNA. The second component of effective targeted gene editing is a single guide RNA (sgRNA) carrying a scaffold sequence which enables its anchoring to Cas9 and a 20 base pair spacer sequence complementary to the target gene and adjacent to the PAM sequence. This sgRNA guides the CRISPR/Cas9 complex to its intended genomic location. The editing system then relies on either of two endogenous DNA repair pathways: non-homologous end-joining (NHEJ) or homology-directed repair (HDR) ( Figure 2 ). NHEJ occurs much more frequently in most cell types and involves random insertion and deletion of base pairs, or indels, at the cut site. This error-prone mechanism usually results in frameshift mutations, often creating a premature stop codon and/or a non-functional polypeptide. This pathway has been particularly useful in genetic knock-out experiments and functional genomic CRISPR screens, but it can also be useful in the clinic in the context where gene disruption provides a therapeutic opportunity. The other pathway, which is especially appealing to exploit for clinical purposes, is the error-free HDR pathway. This pathway involves using the homologous region of the unedited DNA strand as a template to correct the damaged DNA, resulting in error-free repair. Experimentally, this pathway can be exploited by providing an exogenous donor template with the CRISPR/Cas9 machinery to facilitate the desired edit into the genome ( 30 ).

CRISPR/Cas9 mediated gene editing. Cas9 in complex with the sgRNA targets the respective gene and creates DSBs near the PAM region. DNA damage repair proceeds either through the NHEJ pathway or HDR. In the NHEJ pathway, random insertions and deletions (indels) are introduced at the cut side and ligated resulting in error-prone repair. In the HDR pathway, the homologous chromosomal DNA serves as a template for the damaged DNA during repair, resulting in error-free repair.

Limitations and Advancements of CRISPR/Cas9

Off-target effects.

A major concern for implementing CRISPR/Cas9 for gene therapy is the relatively high frequency of off-target effects (OTEs), which have been observed at a frequency of ≥50% ( 31 ). Current attempts at addressing this concern include engineered Cas9 variants that exhibit reduced OTE and optimizing guide designs. One strategy that minimizes OTEs utilizes Cas9 nickase (Cas9n), a variant that induces single-stranded breaks (SSBs), in combination with an sgRNA pair targeting both strands of the DNA at the intended location to produce the DSB ( 32 ). Researchers have also developed Cas9 variants that are specifically engineered to reduce OTEs while maintaining editing efficacy ( Table 1 ). SpCas9-HF1 is one of these high-fidelity variants that exploits the “excess-energy” model which proposes that there is an excess affinity between Cas9 and target DNA which may be enabling OTEs. By introducing mutations to 4 residues involved in direct hydrogen bonding between Cas9 and the phosphate backbone of the target DNA, SpCas9-HF1 has been shown to possess no detectable off-target activity in comparison to wildtype SpCas9 ( 35 ). Other Cas9 variants that have been developed include evoCas9 and HiFiCas9, both of which contain altered amino acid residues in the Rec3 domain which is involved in nucleotide recognition. Desensitizing the Rec3 domain increases the dependence on specificity for the DNA:RNA heteroduplex to induce DSBs, thereby reducing OTEs while maintaining editing efficacy ( 38 , 39 ). One of the more recent developments is the Cas9_R63A/Q768A variant, in which the R63A mutation destabilizes R-loop formation in the presence of mismatches and Q768A mutation increases sensitivity to PAM-distal mismatches ( 49 ). Despite the different strategies, the rational for generating many Cas9 variants with reduced OTEs has been to ultimately reduce general Cas9 and DNA interactions and give a stronger role for the DNA:RNA heteroduplex in facilitating the edits.

Cas9 variants.

Optimizing guide designs can also reduce the frequency of OTEs ( 31 ). Many features in an sgRNA determine specificity including the seed sequence (a 10–12 bp region proximal to PAM on 3′ of spacer sequence) ( 29 , 53 ), GC content ( 54 , 55 ), and modifications such as 5′ truncation of the sgRNA ( 56 ). Several platforms have also been designed to provide optimized guide sequences against target genes, including E-Crisp ( 31 , 57 ), CRISPR-design, CasOFFinder, and others ( 31 ). However, many of these tools are designed based on computational algorithms with varying parameters or rely on phenotypic screens that may be specific to cell types and genomes, generating appreciable noise and lack of generalizability across different experimental setups ( 58 , 59 ). Recently, an additional guide design tool named sgDesigner was developed that addressed these limitations by employing a novel plasmid library in silico that contained both the sgRNA and the target site within the same construct. This allowed collecting Cas9 editing efficiency data in an intrinsic manner and establish a new training dataset that avoids the biases introduced through other models. Furthermore, a comparative performance evaluation to predict sgRNA efficiency of sgDesigner with 3 other commonly used tools (Doench Rule Set 2, Sequence Scan for CRISPR and DeepCRISPR) revealed that sgDesigner outperformed all 3 designer tools in 6 independent datasets, suggesting that sgDesigner may be a more robust and generalizable platform ( 60 ).

Protospacer Adjacent Motif Requirement

An additional limitation of the technology is the requirement for a PAM near the target site. Cas9 from the bacteria Streptococcus pyogenes (SpCas9) is one of the most extensively used Cas9s with a relatively short canonical PAM recognition site: 5′NGG3′, where N is any nucleotide. However, SpCas9 is relatively large and difficult to package into AAV vectors ( 61 , 62 ), the most common delivery vehicle for gene therapy. Staphylococcus aureus Cas9 (SaCas9) is a smaller ortholog that can be packaged more easily in AAV vectors but has a longer PAM sequence: 5′NNGRRT3′ or 5′NNGRR(N)3′, where R is any purine, which further narrows the window of therapeutic targeting sites. Engineered SaCas9 variants have been made, such as KKH SaCas9, which recognizes a 5′NNNRRT3′ PAM, broadening the human targeting sites by 2- to 4-fold. OTEs, however, are observed with frequencies similar to wildtype SaCas9 and need to be considered in designing any therapeutic application ( 33 ). Several other variants of SpCas9 have also been engineered for broadening the gene target window including SpCas9-NG, which recognizes a minimal NG PAM ( 44 ) and xCas9, which recognizes a broad range of PAM including NG, GAA, and GAT ( 43 ). A side by side comparison of both variants revealed that while SpCas9-NG had a broader PAM recognition, xCas9 had the lowest OTE in human cells ( 63 ). Another Cas9 ortholog from the bacteria Streptococcus canis , ScCas9, has been recently characterized with a minimal PAM specificity of 5′NNG3′ and an 89.2% sequence homology to SpCas9 and comparable editing efficiency to SpCas9 in both bacterial and human cells ( 52 ). The most recent development is a variant of SpCas9 named SpRY that has been engineered to be nearly PAMless, recognizing minimal NRN > NYN PAMs. This new variant can potentially edit any gene independent of a PAM requirement, and hence can be used therapeutically against several genetic diseases ( 47 ).

Alternatively, RNA-targeting Cas9 variants have been developed which also broaden the gene targeting spectrum by mitigating PAM requirement restrictions. S. pyogenese Cas9 (SpyCas9) can be manipulated to target RNA by providing a short oligonucleotide with a PAM sequence, known as a PAMmer ( 64 , 65 ), and thus eliminates the need for a PAM site within the target region. Other subsets of Cas enzymes have also been discovered that naturally target RNA independent of a PAM, such as Cas13d. Upon further engineering of this effector, CasRx was developed for efficient RNA-guided RNA targeting in human cells ( 66 , 67 ). Although RNA-targeting CRISPR advances provide a therapeutic opportunity without the risk of DNA-damage toxicity, they exclude the potential for editing a permanent correction into the genome.

DNA-Damage Toxicity

CRISPR-induced DSBs often trigger apoptosis rather than the intended gene edit ( 68 ). Further safety concerns were revealed when using this tool in human pluripotent stem cells (hPSCs) which demonstrated that p53 activation in response to the toxic DSBs introduced by CRISPR often triggers subsequent apoptosis ( 69 ). Thus, successful CRISPR edits are more likely to occur in p53 suppressed cells, resulting in a bias toward selection for oncogenic cell survival ( 70 ). In addition, large deletions spanning kilobases and complex rearrangements as unintended consequences of on-target activity have been reported in several instances ( 71 , 72 ), highlighting a major safety issue for clinical applications of DSB-inducing CRISPR therapy. Other variations of Cas9, such as catalytically inactive endonuclease dead Cas9 (dCas9) in which the nuclease domains are deactivated, may provide therapeutic utility while mitigating the risks of DSBs ( 73 ). dCas9 can transiently manipulate expression of specific genes without introducing DSBs through fusion of transcriptional activating or repressing domains or proteins to the DNA-binding effector ( 74 ). Other variants such as Cas9n can also be considered, which induces SSBs rather than DSBs. Further modifications of these Cas9 variants has led to the development of base editors and prime editors, a key innovation for safe therapeutic application of CRISPR technology (see Precision Gene Editing With CRISPR section).

Immunotoxicity

In addition to technical limitations, CRISPR/Cas9, like traditional gene therapy, still raises concerns for immunogenic toxicity. Charlesworth et al. showed that more than half of the human subjects in their study possessed preexisting anti-Cas9 antibodies against the most commonly used bacterial orthologs, SaCas9 and SpCas9 ( 75 ). Furthermore, AAV vectors are also widely used to deliver CRISPR components for gene therapy. To this end, several Cas9 orthologs and AAV serotypes were tested based on sequence similarities and predicted binding strength to MHC class I and class II to screen for immune orthologs that can be used for safe repeated administration of AAV-CRISPR gene therapy. Although no two AAV serotypes were found to completely circumvent immune recognition, the study verified 3 Cas9 orthologs [SpCas9, SaCas9, and Campylobacter jejuni Cas9 (CjCas9)] which showed robust editing efficiency and tolerated repeated administration due to reduced immunogenic toxicity in mice immunized against AAV and Cas9 ( 76 ). A major caveat is pre-existing immunity in humans against 2 of these orthologs—SpCas9 and SaCas9, leaving CjCas9 as the only current option for this cohort of patients. However, this ortholog has not been well-studied in comparison to the other 2 orthologs and will need further investigation to provide evidence for its safety and efficacy for clinical use. Future studies may also identify other Cas9 immune-orthogonal orthologs for safe repeated gene therapy.

Precision Gene Editing With CRISPR

Precise-genome editing is essential for prospects of CRISPR gene therapy. Although HDR pathways can facilitate a desired edit, its low efficiency renders its utility for precise gene editing for clinical intervention highly limiting, with NHEJ as the default pathway human cells take for repair. Enhancement of HDR efficiency has been achieved via suppression of the NHEJ pathway through chemical inhibition of key NHEJ modulating enzymes such as Ku ( 77 ), DNA ligase IV ( 78 ), and DNA-dependent protein kinases (DNA-PKcs) ( 79 ). Other strategies that improve HDR efficiency include using single-stranded oligodeoxynucleotide (ssODN) template, which contains the homology arms to facilitate recombination and the desired edit sequence, instead of double-stranded DNA (dsDNA). Rationally designed ssODN templates with optimized length complementarity have been shown to increase HDR rates up to 60% in human cells for single nucleotide substitution ( 80 ). Furthermore, cell cycle stage plays a key role in determining the DNA-damage repair pathway a cell may take. HDR events are generally restricted to late S and G2 phases of the cell cycle, given the availability of the sister chromatid to serve as a template at these stages, whereas NHEJ predominates the G1, S, and G2 phases ( 81 ). Pharmacological arrest at the S phase with aphidicolin increased HDR frequency in HEK293T with Cas9-guide ribonucleoprotein (RNP) delivery. Interestingly, cell arrest in the M phase using nocodazole with low concentrations of the Cas9-guide RNP complex yielded higher frequencies of HDR events in these cells, reaching a maximum frequency of up to 31% ( 82 ). Although HDR is considered to be restricted to mitotic cells, a recent study revealed that the CRISPR/Cas9 editing can achieve HDR in mature postmitotic neurons. Nishiyama et al. successfully edited the CaMKIIα locus through HDR in postmitotic hippocampal neurons of adult mice in vitro using an AAV delivered Cas9, guide RNA, and donor template in the CaMKIIα locus, which achieved successful HDR-mediated edits in ~30% of infected cells. Although HDR efficiency was dose-dependent on AAV delivered HDR machinery and off-target activity was not monitored, this study demonstrated CRISPR's potential utility for translational neuroscience after further developments ( 83 ). To further exploit cell-cycle stage control as a means to favor templated repair, Cas9 conjugation to a part of Geminin, a substrate for G1 proteosome degradation, can limit Cas9 expression to S, G2, and M stages. This strategy was shown to facilitate HDR events while mitigating undesired NHEJ edits in human immortalized and stem cells ( 84 , 85 ). A more recent strategy combined a chemically modified Cas9 to the ssODN donor or a DNA adaptor that recruits the donor template, either of which improved HDR efficiency by localizing the donor template near the cleavage site ( 86 ). Despite these advancements, HDR is still achieved at a relatively low efficiency in eukaryotic cells and use of relatively harmful agents in cells such as NHEJ chemical inhibitors may not be ideal in a clinical setting.

A recent advancement that allows precision gene editing independent of exploiting DNA damage response mechanisms is the CRISPR base editing (BE) system. In this system, a catalytically inactive dead Cas9 (dCas9) is conjugated to deaminase, which can catalyze the conversion of nucleotides via deamination. For increased editing efficiency, Cas9 nickase (Cas9n) fused with deaminase is recently being more utilized over dCas9 for base editing, as the nicks created in a single strand of DNA induce higher editing efficiency. Currently, the two types of CRISPR base editors are cytidine base editors (CBEs) and adenosine base editors (ABEs). CBEs catalyze the conversion of cytidine to uridine, which becomes thymine after DNA replication. ABEs catalyze the conversion of adenosine to inosine which becomes guanine after DNA replication ( 87 ). Base editors provide a means to edit single nucleotides without running the risk of causing DSB-induced toxicity. However, base editors are limited to “A to T” and “C to G” conversions, narrowing its scope for single-base gene edition to only these bases. In addition, base editors still face some of the same challenges as the previously described CRISPR systems, including OTEs, more so with CBEs than ABEs ( 88 , 89 ) and packaging constraints, namely in AAV vectors due to the large size of base editors ( 90 ). Furthermore, the editing window for base editors are limited to a narrow range of a few bases upstream of the PAM ( 90 ). More recently, prime editing has been developed as a strategy to edit the genome to insert a desired stretch of edits without inducing DSBs ( 91 ). This technology combines fusion of Cas9n with a reverse transcriptase and a prime editing guide RNA (pegRNA), which contains sgRNA sequence, primer binding site (PBS), and an RNA template encoding the desired edit on the 3′ end. Prime editors use Cas9n to nick one strand of the DNA and insert the desired edit via reverse transcription of the RNA template. The synthesized edit is incorporated into the genome and the unedited strand is cleaved and repaired to match the inserted edit. With an optimized delivery system in place, base editors and primer editors can open the door for precision gene editing to correct and potentially cure a multitude of genetic diseases ( Figure 3 ).

Precise Gene Editing. (A) CRISPR/Cas9-HDR. Cas9 induces a DSB. The exogenous ssODN carrying the sequence for the desired edit and homology arms is used as a template for HDR-mediated gene modification. (B) Base Editor. dCas9 or Cas9n is tethered to the catalytic portion of a deaminase. Cytosine deaminase catalyzes the formation of uridine from cytosine. DNA mismatch repair mechanisms or DNA replication yield an C:G to T:A single nucleotide base edit. Adenosine deaminase catalyzes the formation of inosine from adenosine. DNA mismatch repair mechanisms or DNA replication yield an A:T to G:C single nucleotide base edit. (C) Prime Editor. Cas9n is tethered to the catalytic portion of reverse transcriptase. The prime editor system uses pegRNA, which contains the guide spacer sequence, reverse transcriptase primer, which includes the sequence for the desired edit and a primer binding site (PBS). PBS hybridizes with the complementary region of the DNA and reverse transcriptase transcribes new DNA carrying the desired edit. After cleavage of the resultant 5′ flap and ligation, DNA repair mechanisms correct the unedited strand to match the edited strand. HDR, homology directed repair. DSB, double stranded break; SSB, single-stranded break; ssODN, single-stranded oligodeoxynucleotide.

Delivery of CRISPR Gene Therapy

The delivery modality of CRISPR tools greatly influences its safety and therapeutic efficacy. While traditional gene therapy utilizing viruses have been scrutinized for the risk of immunotoxicity and insertional oncogenesis, AAV vectors remain a key delivery vehicle for CRISPR gene therapy and continues to be extensively used for its high efficiency of delivery ( 92 ). The CRISPR toolkit can be packaged as plasmid DNA encoding its components, including Cas9 and gRNA, or can be delivered as mRNA of Cas9 and gRNA. Nucleic acids of CRISPR can be packaged in AAV vectors for delivery or introduced to target cells via electroporation/nucleofection or microinjection, with the latter methods averting virus-associated risks. However, microinjection can be technically challenging and is only suited for ex vivo delivery. Electroporation is also largely used for ex vivo but can be used in vivo for certain target tissues ( 93 ). However, high-voltage shock needed to permeabilize cell membranes via electroporation can be toxic and can lead to permanent permeabilization of treated cells ( 94 ). In addition to viral toxicity, AAV delivery of CRISPR components yields longevity of expression, leading to greater incidence of OTEs. Alternatively, delivery of the Cas9 protein and gRNA as RNP complexes has reduced OTEs while maintained editing efficacy, owing to its transient expression and rapid clearance in the cell ( 95 ).

Once the delivery modality is selected, CRISPR/Cas9 edits can be facilitated either ex vivo where cells are genetically modified outside of the patient and reintroduced back, or in vivo with delivery of the CRISPR components directly into the patient where cells are edited ( Figure 4 ). Both systems pose their own set of advantages and challenges. Advantages for ex vivo delivery include greater safety since patients are not exposed to the gene altering tool, technical feasibility, and tighter quality control of the edited cells. However, challenges to this method include survival and retention of in vivo function of cells outside the patient after genetic manipulation and extensive culture in vitro . Also, an adequate supply of cells is needed for efficient re-engraftment. These conditions limit this method to certain cell types that can survive and be expanded in culture, such as hematopoietic stem and progenitor cells (HSPCs) ( 96 ) and T cells ( 97 ).

Delivery of CRISPR Therapy. Nucleic acids encoding CRISPR/Cas9 or its RNP complex can be packaged into delivery vehicles. Once packaged, edits can be facilitated either ex vivo or in vivo . Ex vivo editing involves extraction of target cells from the patient, cell culture, and expansion in vitro , delivery of the CRISPR components to yield the desired edits, selection, and expansion of edited cells, and finally reintroduction of therapeutic edited cells into the patient. In vivo editing can be systemically delivered via intravenous infusions to the patient, where the CRISPR cargo travels through the bloodstream via arteries leading to the target tissue, or locally delivered with injections directly to target tissue. Once delivered, the edits are facilitated in vivo to provide therapeutic benefit.

While ex vivo gene therapy has provided therapeutic benefit for hematological disorders and cancer immunotherapy, many tissue types are not suited for this method, severely limiting its therapeutic utility for other genetic diseases. in vivo manipulation is thus needed to expand CRISPR's utility to treat a broader range of genetic diseases, such as Duchenne muscular dystrophy (DMD) ( 98 ) and hereditary tyrosinemia ( 99 ). CRISPR components can be delivered in vivo systemically through intravenous injections or can be locally injected to specific tissues ( Figure 4 ). With systemic delivery, the CRISPR components and its vehicle are introduced into the circulatory system where expression of the gene editing toolkit can be controlled to target specific organs via tissue-specific promoters ( 100 ). However, challenges of in vivo delivery include degradation by circulating proteases or nucleases, opsonization by opsonins, or clearance by the mononuclear phagocyte system (MPS). Furthermore, the cargo must reach the target tissue and bypass the vascular endothelium, which are often tightly connected by cell-cell junctions ( 101 ), preventing accessibility to larger delivery vehicles (>1 nm diameter). Additionally, once the cargo has reached the target cells, they must be internalized, which is generally facilitated through endocytosis where they can be transported and degraded by lysosomal enzymes ( 102 ). In addition, localization of the editing machinery near the point of injection can result in uneven distribution of the edited cell repertoire within the tissue, which may result in suboptimal therapeutic outcomes ( 102 ). While advancements are continuing to refine delivery techniques, the current systems have allowed CRISPR gene therapy to be used in the clinic.

Biological Intervention of CRISPR/Cas9 in Clinical Trials

Cancer immunotherapy.

The first CRISPR Phase 1 clinical trial in the US opened in 2018 with the intent to use CRISPR/Cas9 to edit autologous T cells for cancer immunotherapy against several cancers with relapsed tumors and no further curative treatment options. These include multiple myeloma, melanoma, synovial sarcoma and myxoid/round cell liposarcoma. This trial was approved by the United States Food and Drug Administration (FDA) after careful consideration of the risk to benefit ratios of this first application of CRISPR gene therapy into the clinic. During this trial, T lymphocytes were collected from the patients' blood and ex vivo engineered with CRISPR/Cas9 to knockout the α and β chains of the endogenous T cell receptor (TCR), which recognizes a specific antigen to mediate an immune response, and the programmed cell death-1 (PD-1) protein, which attenuates immune response. The cells were then transduced with lentivirus to deliver a gene encoding a TCR specific for a NY-ESO-1 antigen, which has been shown to be highly upregulated in the relapsed tumors and thus can serve as a therapeutic target. Since then, many trials have opened for CRISPR-mediated cancer immunotherapy and is currently the most employed strategy for CRISPR gene therapy ( Table 2 ). A trial implementing this strategy using other tools had already been conducted in both pre-clinical and clinical settings, but this was the first time CRISPR/Cas9 was used to generate the genetically modified T cells ( 97 ). The moderate transition of switching only the tool used for an already approved therapeutic strategy may have been key to paving the road for using CRISPR's novel abilities for gene manipulation, such as targeted gene disruption.

Biological intervention of CRISPR gene therapy in clinical trials.

Gene Disruption

The first clinical trial in the US using CRISPR to catalyze gene disruption for therapeutic benefit were for patients with sickle-cell anemia (SCD) and later β-thalassemia, by Vertex Pharmaceuticals and CRISPR Therapeutics. This therapy, named CTX001, increases fetal hemoglobin (HbF) levels, which can occupy one or two of four hemoglobin binding pockets on erythrocytes and thereby provides clinical benefit for major β-hemoglobin diseases such as SCD and β-thalassemia ( 103 ). The trial involved collecting autologous hematopoietic stem and progenitor cells from peripheral blood and using CRISPR/Cas9 to disrupt the intronic erythroid-specific enhancer for the BCL11A gene ( {"type":"clinical-trial","attrs":{"text":"NCT03745287","term_id":"NCT03745287"}} NCT03745287 ) as disruption of this gene increases HbF expression ( 104 – 106 ). Genetically modified hematopoietic stem cells with BCL11A disruption are delivered by IV infusion after myeloablative conditioning with busulfan to destroy unedited hematopoietic stem cells in the bone marrow. Preliminary findings from two patients receiving this treatment seem promising. One SCD patient was reported to have 46.6% HbF and 94.7% erythrocytes expressing HbF after 4 months of CTX001 transfusions and one β-thalassemia patient is expressing 10.1 g/dL HbF out of 11.9 g/dL total hemoglobin, and 99.8% erythrocytes expressing HbF after 9 months of the therapy. Results from the clinical trial that has opened for this therapy ( {"type":"clinical-trial","attrs":{"text":"NCT04208529","term_id":"NCT04208529"}} NCT04208529 ) to assess the long-term risks and benefits of CTX001 will dictate whether this approach can provide a novel therapeutic opportunity for a disease that otherwise has limited treatment options.

In vivo CRISPR Gene Therapy

While the aforementioned trials rely on ex vivo editing and subsequent therapy with modified cells, in vivo approaches have been less extensively employed. An exciting step forward with CRISPR gene therapy has been recently launched with a clinical trial using in vivo delivery of CRISPR/Cas9 for the first time in patients. While in vivo editing has been largely limited by inadequate accessibility to the target tissue, a few organs, such as the eye, are accessible. Leber congenital amaurosis (LCA) is a debilitating monogenic disease that results in childhood blindness caused by a bi-allelic loss-of-function mutation in the CEP290 gene, with no treatment options. This therapy, named EDIT-101, delivers CRISPR/Cas9 directly into the retina of LCA patients specifically with the intronic IVS26 mutation, which drives aberrant splicing resulting in a non-functional protein. The therapy uses an AAV5 vector to deliver nucleic acid instructions for Staphylococcus aureus Cas9 and two guides targeting the ends of the CEP290 locus containing the IVS26 mutation. The DSB induced by Cas9 and both guides result in either a deletion or inversion of the IVS26 intronic region, thus preventing the aberrant splicing caused by the genetic mutation and enabling subsequent translation of the functional protein ( 107 ). Potential immunotoxicity or OTEs arising from nucleic acid viral delivery will have to be closely monitored. Nonetheless, a possibly curative medicine for genetic blindness using an in vivo approach marks an important advancement for CRISPR gene therapy.

CRISPR Editing in Human Embryos and Ethical Considerations

While somatic editing for CRISPR therapy has been permitted after careful consideration, human germline editing for therapeutic intent remains highly controversial. With somatic edition, any potential risk would be contained within the individual after informed consent to partake in the therapy. Embryonic editing not only removes autonomy in the decision-making process of the later born individuals, but also allows unforeseen and permanent side effects to pass down through generations. This very power warrants proceeding with caution to prevent major setbacks as witnessed by traditional gene therapy. However, a controversial CRISPR trial in human embryos led by Jiankui He may have already breached the ethical standards set in place for such trials. This pilot study involved genetic engineering of the C-C chemokine receptor type 5 ( CCR5 ) gene in human embryos, with the intention of conferring HIV-resistance, as seen by a naturally occurring CCR5 Δ 32 mutation in a few individuals ( 108 ). However, based on the limited evidence, CRISPR/Cas9 was likely used to target this gene, but rather than replicate the naturally observed and beneficial 32-base deletion, the edits merely induced DSBs at one end of the deletion, allowing NHEJ to repair the damaged DNA while introducing random, uncharacterized mutations. Thus, it is unknown whether the resultant protein will function similarly to the naturally occurring CCR5 Δ 32 protein and confer HIV resistance. In addition, only one of the two embryos, termed with the pseudonym Nana, had successful edits in both copies of the CCR5 gene, whereas the other embryo, with pseudonym Lulu, had successful editing in only one copy. Despite these findings, both embryos were implanted back into their mother, knowing that the HIV-resistance will be questionable in Nana and non-existent in Lulu ( 109 , 110 ).

Furthermore, recent studies have shown that the mechanism for infection of some variants of the highly mutable HIV virus may heavily rely on the C-X-C chemokine receptor type 4 ( CXCR4 ) co-receptor ( 108 , 111 ). With no attempts at editing CXCR4 , this adds yet another layer of skepticism toward achieving HIV resistance by this strategy. In addition, OTEs, particularly over the lifetime of an individual, remain a major concern for applying this technology in humans. The recent advances in the editing tool to limit OTEs, such as using high fidelity Cas9 variants, has not been exploited. Furthermore, the rationale for selecting HIV prevention for the first use of CRISPR in implanted human embryos contributes to the poor risk to benefit ratio of this study, considering HIV patients can live long, healthy lives on a drug regimen. A more appropriate first attempt would have been to employ this technology for a more severe disease. For example, correction of the MYBPC3 gene is arguably a better target for embryonic gene editing, as mutations in MYBPC3 can cause hypertrophic cardiomyopathy (HCM), a heart condition responsible for most sudden cardiac deaths in people under the age of 30. Gene correction for this pathological mutation was achieved recently for the first time in the US in viable human embryos using the HDR-mediated CRISPR/Cas9 system. However, these embryos were edited for basic research purposes and not intended for implantation. In this study, sperm carrying the pathogenic MYBPC3 mutation and the CRISPR/Cas9 machinery as an RNP complex were microinjected into healthy donor oocytes arrested at MII, achieving 72.4% homozygous wildtype embryos as opposed to 47.4% in untreated embryos. The HDR-mediated gene correction was observed at considerably high frequencies with no detectable OTEs in selected blastomeres, likely owing to the direct microinjection delivery of the RNP complex in the early zygote. Interestingly, the maternal wildtype DNA was used preferentially for templated repair over the provided exogenous ssODN template ( 112 ). While evidence for gene correction was promising, NHEJ mediated DNA repair was still observed in many embryos, highlighting the need to improve HDR efficiency before clinical application can be considered. Although strategies have been developed to improve HDR, such as chemical inhibitors of NHEJ ( 77 – 79 ), such techniques may have varying outcomes in embryonic cells and side effects that may arise from treatment needs to be investigated. Germline gene editing will remain to be ethically unfavorable at its current state and its discussions may not be considered until sufficient long-term studies of the ongoing somatic CRISPR therapy clinical trials are evaluated.

Potential for CRISPR Therapeutics During COVID-19 Pandemic

The rapidly advancing CRISPR technology may provide aid during our rapidly evolving times. The recent outbreak of a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic ( 113 ). These pressing times call for an urgent response to develop quick and efficient testing tools and treatment options for coronavirus disease 2019 (COVID-19) patients. Currently available methods for testing are relatively time consuming with suboptimal accuracy and sensitivity ( 114 ). The two predominant testing methods are molecular testing or serological testing. The US Centers for Disease Control and Prevention (CDC) has developed a real-time RT-PCR assay for molecular testing for the presence of viral RNA to detect COVID-19 ( 115 ). However, this assay has a roughly ~30% false negative rate ( 116 , 117 ) with the turnaround time of several hours to >24 h. Serological testing methods are much more rapid but lack the ability to detect acute respiratory infection since antibodies used to detect infection can take several days or weeks to develop.

Recently, a CRISPR Cas12-based assay named SARS-CoV-2 DETECTR has been developed for detection of COVID-19 with a short turnaround time of about 40 min and a 95% reported accuracy. The assay involves RNA extraction followed by reverse transcription and simultaneous isothermal amplification using the RT-LAMP method. Cas12 and a guide RNA against regions of the N (nucleoprotein) gene and E (envelope) gene of SARS-CoV-2 are then targeted, which can be visualized by cleavage of a fluorescent reporter molecule. The assay also includes a laminar flow strip for a visual readout, where a single band close to where the sample was applied indicates a negative test and 2 higher bands or a single higher band would indicate cleavage of the fluorescent probe and hence positive for SARS-CoV-2 ( 118 ).

In addition to CRISPR's diagnostic utility, CRISPR may provide therapeutic options for COVID-19 patients. The recently discovered Cas13 is an RNA-guided RNA-targeting endonuclease may serve as a potential therapeutic tool against COVID-19. PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells) has been developed, which utilizes the Ruminococcus flavefaciens derived VI-D CRISPR-Cas13d variant, selected for its small size facilitating easier packaging in viral vehicles, high specificity, and strong catalytic activity in human cells. This technique was developed to simultaneously target multiple regions for RNA degradation, opening the door for a much-needed pan-coronavirus targeting strategy, given the evidence suggesting relatively high mutation and recombination rates of SARS-CoV-2 ( 119 ). With these advances, the CRISPR/Cas machinery may again be implemented to serve its original purpose as a virus-battling system to provide aid during this pandemic.

The birth of gene therapy as a therapeutic avenue began with the repurposing of viruses for transgene delivery to patients with genetic diseases. Gene therapy enjoyed an initial phase of excitement, until the recognition of immediate and delayed adverse effects resulted in death and caused a major setback. More recently, the discovery and development of CRISPR/Cas9 has re-opened a door for gene therapy and changed the way scientists can approach a genetic aberration—by fixing a non-functional gene rather than replacing it entirely, or by disrupting an aberrant pathogenic gene. CRISPR/Cas9 provides extensive opportunities for programmable gene editing and can become a powerful asset for modern medicine. However, lessons learned from traditional gene therapy should prompt greater caution in moving forward with CRISPR systems to avoid adverse events and setbacks to the development of what may be a unique clinically beneficial technology. A failure to take these lessons into account may provoke further backlash against CRISPR/Cas9 development and slow down progression toward attaining potentially curative gene editing technologies.

Although CRISPR editing in humans remains a highly debated and controversial topic, a few Regulatory Affairs Certification (RAC)-reviewed and FDA-approved CRISPR gene therapy trials have opened after thorough consideration of the risk to benefit ratios. These first few approved trials, currently in Phase I/II, are only for patients with severe diseases, such as cancers or debilitating monogenic diseases. The outcomes of these trials will dictate how rapidly we consider using this system to treat less severe diseases, as the risks of the technology are better understood. A concern remains whether normalizing CRISPR/Cas9 editing for less debilitating diseases may act as a gateway for human genome editing for non-medical purposes, such as altering genes in embryos to create offspring with certain aesthetic traits. This fear of unnatural selection for unethical reasons has likely become more tangible in the public's view with the strong media attention of the edited “CRISPR babies.” The lasting effects of that trial and outcomes of the approved clinical trials will greatly influence CRISPR's future in gene therapy and begin to answer the key questions we must consider as we further explore this technology. These key questions include how to avoid the mistakes of the past, who should decide CRISPR's therapeutic future, and how the ethical boundaries of its applications should best be drawn.

Author Contributions

FU researched and drafted the article. TS and CR supervised the content. All authors wrote, reviewed, and edited the manuscript before submission.

Conflict of Interest

CR has consulted regarding oncology drug development with AbbVie, Amgen, Ascentage, Astra Zeneca, Celgene, Daiichi Sankyo, Genentech/Roche, Ipsen, Loxo, and Pharmar, and is on the scientific advisory boards of Harpoon Therapeutics and Bridge Medicines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors would like to thank Ms. Emily Costa, Dr. Alvaro Quintanal Villalonga, and Dr. Rebecca Caesar for their excellent assistance with editing the review.

Funding. This work was supported by grants from the US National Institutes of Health, including U24CA213274 and R01CA197936 (CR); Parker Institute of Cancer Immunotherapy grant (TS).

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Gene therapy provision would make life-saving treatment more accessible and equitable.

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Celia Grace Hamlett was 4 when she was diagnosed with metachromatic leukodystrophy (MLD), a rare, genetic neurological disease that leads to severe disability and eventual death. The devastating news thrust her family into a quest for treatment, a journey that led them from their Alabama home to our team at M Health Fairview Masonic Children's Hospital .

While the Hamletts grappled with this diagnosis, we lobbied the U.S. Food and Drug Administration (FDA) for special permission to perform the experimental procedure. After multiple hurdles with insurance coverage, Celia Grace became the first MLD patient in the U.S. to be treated with gene therapy. As the process unfolded, a local documentary film crew picked up cameras to capture this turning point in modern medicine.

After three years of filming and about a month after the FDA approved the same therapy that Celia Grace received in 2021, "Sequencing Hope" premiered at the Minneapolis-St. Paul International Film Festival. It documented Celia Grace's path to treatment and her life in the years since. For Celia Grace and many others facing similar challenges, gene therapy represents more than just treatment — it offers the possibility of a future once deemed impossible.

As physicians and researchers with M Health Fairview and the University of Minnesota Medical School, we have devoted our careers to exploring treatments and cures for conditions such as inherited neurological disorders, enzyme deficiencies and sickle cell disease. Through rigorous clinical trials, we have witnessed firsthand the life-changing potential for gene therapies to cure people with conditions who previously lacked safe and effective treatment options. We are deeply concerned, however, that without comprehensive payment coverage from Medicaid and private insurers, these groundbreaking discoveries will remain out of reach for many.

The steep toll of gene therapies, ranging from $2 million to more than $4 million, poses significant challenges for health care delivery systems, which are not designed to account for the multimillion-dollar upfront costs of these therapies. Without comprehensive coverage from Medicaid and private insurers, equitable access to transformative gene therapies is becoming a greater obstacle than the development of the therapies themselves.

At the core of this issue lies the imperative of equitable access to these revolutionary treatments. Communities of color, disproportionately affected by diseases like sickle cell disease, may bear the brunt of inadequate access to care. As the medical community works to explore the promise of gene therapy, private and public payers and drug manufacturers must work with providers who are familiar with these conditions to develop a payment model that will ensure equitable access to this lifesaving care.

Minnesota legislators have the power to address these disparities by acting this session on legislation that authorizes the state to provide the required reimbursement for gene therapy products delivered within hospital inpatient settings. This is a critical step that other states have taken and represents a lifeline for those in desperate need. In Minnesota, where approximately 1,000 individuals are affected by sickle cell disease, the need for equitable access to these lifesaving treatments cannot be overstated.

Passing legislation to cover the costs of gene therapy also makes sound economic sense. While the initial costs may seem prohibitive, data suggests that the long-term savings in health care expenditures are substantial.

Most important, the impact on patients' lives is immeasurable. By embracing these therapies, we can give more Minnesotans the lifesaving opportunities that saved the life of Celia Grace. We urge our legislators to pass the necessary provisions to ensure that no one is left behind in the march toward progress.

Paul Orchard, Roy Kao and Ashish Gupta are physicians at M Health Fairview who specialize in treating rare diseases. They also research and educate the next generation of medical professionals at the University of Minnesota Medical School. The gene therapy coverage provision was included in the Minnesota Senate's health finance omnibus budget bill. But it was not in the House version, which was passed May 9 . A conference committee is now reconciling the two bills.

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