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Key Points Inherited diseases still represent a substantial burden to modern societies. The identification of causative gene defects for several of these diseases facilitated the development of gene-based therapeutic strategies. Over the past few years, clinical translation of bench research resulted in the first examples of therapeutic success for the field of gene transfer. Adeno-associated virus (AAV) vectors are among the most suitable tools for in vivo gene delivery given their superior efficiency of transduction of several tissues, including liver, muscle and nervous tissue. Preclinical studies with these vectors have shown stable transgene expression and long-term correction of disease phenotype with little to no toxicity in small and large animal models of disease. In humans, some of the most promising results were obtained when AAV was used to transfer therapeutic genes to the retina for the treatment of congenital blindness caused by retinal pigment epithelium-specific protein 65kDa (RPE65) deficiency, to the liver to correct the bleeding phenotype of haemophilia B, and to focal areas of the brain to ameliorate the course of Parkinson's disease, an acquired neurodegenerative disease. The clinical experience with AAV vectors also highlighted a number of issues, some of which are not predicted by animal studies. Among them, vector or transgene immunogenicity remains the major obstacle to long-term therapeutic transgene expression. Target-tissue- and disease-specific hurdles will also need to be addressed to expand the scope of success with the AAV gene transfer platform. The eye represents an ideal target tissue for gene transfer as widespread organ transduction can be achieved at low vector doses, and because presentation of a foreign antigen in this compartment is usually not associated with inflammatory responses. Experience with RPE65 deficiency is paving the way for the development of a whole new class of gene therapeutics targeting the retina. The liver is also one of the most attractive targets for gene transfer owing to its unique biosynthetic capabilities and its ability to favour antigen-specific tolerance upon gene transfer. Some exciting results have recently been obtained in the clinic with haemophilia B; overcoming the limit of capsid-driven immune responses promises even greater success. Gene transfer to the central nervous system has also given promising results. Efficient global gene transfer — achieved with novel delivery techniques and/or novel serotypes — will be the key to successfully treating some neurodegenerative diseases, such as lysosomal storage disorders. Efficient delivery techniques are under development to target large areas of muscle using AAV vectors. Hopefully this will help us to reach the levels of transgene expression needed for therapeutic efficacy in some diseases. The identification of the hurdles for clinical translation of AAV gene transfer, along with the development of solutions to overcome these problems, will enable us to provide improved therapeutic options for a range of inherited diseases by fully exploiting the potential of in vivo gene transfer. Recent clinical and preclinical studies have described exciting results using recombinant adeno-associated virus (AAV) vectors for therapeutic gene transfer in genetic disease. This Review explores the potential for using this form of therapy in the context of different tissues and diseases. In vivo gene replacement for the treatment of inherited disease is one of the most compelling concepts in modern medicine. Adeno-associated virus (AAV) vectors have been extensively used for this purpose and have shown therapeutic efficacy in a range of animal models. Successful translation to the clinic was initially slow, but long-term expression of donated genes at therapeutic levels has now been achieved in patients with inherited retinal disorders and haemophilia B. Recent exciting results have raised hopes for the treatment of many other diseases. As we discuss here, the prospects and challenges for AAV gene therapy are to a large extent dependent on the target tissue and the specific disease.

Gene and cell therapy products approved over the past decade in Europe and North America have provided new therapeutic options for single gene disorders and for hematologic malignancies. Lessons learned, and limitations identified, are reviewed.

Nearly 50 years after the concept was first proposed, gene therapy is now considered a promising treatment option for several human diseases. The path to success has been long and tortuous. Serious adverse effects were encountered in early clinical studies, but this fueled basic research that led to safer and more efficient gene transfer vectors. Gene therapy in various forms has produced clinical benefits in patients with blindness, neuromuscular disease, hemophilia, immunodeficiencies, and cancer. Dunbar et al. review the pioneering work that led the gene therapy field to its current state, describe gene-editing technologies that are expected to play a major role in the field's future, and discuss practical challenges in getting these therapies to patients who need them. Science , this issue p. eaan4672 After almost 30 years of promise tempered by setbacks, gene therapies are rapidly becoming a critical component of the therapeutic armamentarium for a variety of inherited and acquired human diseases. Gene therapies for inherited immune disorders, hemophilia, eye and neurodegenerative disorders, and lymphoid cancers recently progressed to approved drug status in the United States and Europe, or are anticipated to receive approval in the near future. In this Review, we discuss milestones in the development of gene therapies, focusing on direct in vivo administration of viral vectors and adoptive transfer of genetically engineered T cells or hematopoietic stem cells. We also discuss emerging genome editing technologies that should further advance the scope and efficacy of gene therapy approaches.

Leber's congenital amaurosis (LCA) is a group of inherited disorders involving retinal degeneration with severe vision loss noted in early infancy. The condition is usually identified through behaviors, including abnormal roving-eye movements (nystagmus). The diagnosis is confirmed by both abnormal electroretinographic responses and pupillary light reflexes. 1 – 4 Most patients with LCA have severe visual impairment throughout childhood; vision deteriorates over time, and patients usually have total blindness by the third or fourth decade of life. 4 There is no treatment for LCA. The LCA2 form of the disease is associated with mutations in RPE65, which encodes a protein requisite for the . . .

Those who have followed the gene-therapy field over the decades may be weary of forward-looking positive statements. However, over the past 3 years, six gene-therapy products have been approved for clinical use. This article describes challenges, risks, and advances in gene-therapy clinical research.

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in “humanized” mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Leber's congenital amaurosis. We assessed the retinal and visual function in 12 patients (aged 8–44 years) with RPE65-associated Leber's congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1·5×10 10 vector genomes), medium (4·8×10 10 vector genomes), or high dose (1·5×10 11 vector genomes) for up to 2 years. AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with ClinicalTrials.gov, number NCT00516477. The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain. Center for Cellular and Molecular Therapeutics at the Children's Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.

Therapeutic gene transfer holds the promise of providing lasting therapies and even cures for diseases that were previously untreatable or for which only temporary or suboptimal treatments were available. For some time, clinical gene therapy was characterized by some impressive but rare examples of successes and also several setbacks. However, effective and long-lasting treatments are now being reported from gene therapy trials at an increasing pace. Positive outcomes have been documented for a wide range of genetic diseases (including hematological, immunological, ocular, and neurodegenerative and metabolic disorders) and several types of cancer. Examples include restoration of vision in blind patients, eradication of blood cancers for which all other treatments had failed, correction of hemoglobinopathies and coagulation factor deficiencies, and restoration of the immune system in children born with primary immune deficiency. To date, about 2,000 clinical trials for various diseases have occurred or are in progress, and many more are in the pipeline. Multiple clinical studies reported successful treatments of pediatric patients. Design of gene therapy vectors and their clinical development are advancing rapidly. This article reviews some of the major successes in clinical gene therapy of recent years.

Formation of pathogenic antibodies is a major problem in replacement therapies for inherited protein deficiencies. For example, antibodies to coagulation factors (‘inhibitors’) seriously complicate treatment of haemophilia. While immune tolerance induction (ITI) protocols have been developed, inhibitors against factor IX (FIX) are difficult to eradicate due to anaphylactic reactions and nephrotic syndrome and thus substantially elevate risks for morbidity and mortality. However, hepatic gene transfer with an adeno‐associated virus (AAV) serotype 8 vector expressing FIX (at levels of ≥4% of normal) rapidly reversed pre‐existing high‐titre inhibitors in haemophilia B mice, eliminated antibody production by B cells, desensitized from anaphylaxis (even if protein therapy was resumed) and provided long‐term correction. High levels of FIX protein suppressed memory B cells and increased Treg induction, indicating direct and indirect mechanisms of suppression of inhibitor formation. Persistent presence of Treg was required to prevent relapse of antibodies. Together, these data suggest that hepatic gene transfer‐based ITI provides a safe and effective alternative to eradicate inhibitors. This strategy may be broadly applicable to reversal of antibodies in different genetic diseases. Graphical Abstract Hepatic adeno‐associated viral factor IX gene transfer rapidly reversed pre‐existing high‐titer inhibitors in hemophilia B mice, eliminated antibody production by B cells, desensitized from anaphylaxis and provided long‐term correction.

Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.