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Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

This viewpoint discusses cost-effectiveness estimates for EtranaDez, a gene therapy for hemophilia B, using the Institute for Clinical and Economic Reviews (ICER) framework for single and short-term therapies (SSTs). EtranaDez offers long-term benefits from a single administration, in contrast to the high costs and frequent dosing required by current factor IX prophylaxis. However, the projected gains in health from EtranaDez are small relative to the cost implications of the therapy, and consequently, how the cost offsets associated with EtranaDez are counted has a substantial impact on assessing its cost-effectiveness. Strategies for assessing cost offsets used in the ICER SST framework include a 50/50 cost-sharing model between the health care system and the manufacturer and a cap of $150,000 annually on health care cost offsets. Results from the standard full cost-offset analysis as reported by ICER depicted EtranaDez as a dominant therapy with substantial cost savings compared with factor IX prophylaxis. However, while considering the ICER SST framework, particularly the $150,000 annual cap scenario, the cost-effectiveness was significantly reduced. The incremental cost-effectiveness ratio varied notably between these scenarios, challenging the conventional perception of value of gene therapy in health care. These cost-sharing scenarios highlight the potential of the ICER SST framework to help curtail inefficient health care spending. In cases in which the cost of existing treatment is exceedingly high, the application of such frameworks would improve efficiency in resource allocation, fostering a balance between incentives for innovation and economic sustainability in managed care systems.

There are more than 10,000 individual rare diseases and most are without therapy. Personalized genetic therapy represents one promising approach for their treatment. We present a road map for individualized treatment of an ultra-rare disease by establishing a gene replacement therapy developed for a single patient with hereditary spastic paraplegia type 50 (SPG50). Through a multicenter collaboration, an adeno-associated virus-based gene therapy product carrying the AP4M1 gene was created and successfully administered intrathecally to a 4-year-old patient within 3 years of diagnosis as part of a single-patient phase 1 trial. Primary endpoints were safety and tolerability, and secondary endpoints evaluated efficacy. At 12 months after dosing, the therapy was well tolerated. No serious adverse events were observed, with minor events, including transient neutropenia and Clostridioides difficile gastroenteritis, experienced but resolved. Preliminary efficacy measures suggest a stabilization of the disease course. Longer follow-up is needed to confirm the safety and provide additional insights on the efficacy of the therapy. Overall, this report supports the safety of gene therapy for SPG50 and provides insights into precision therapy development for rare diseases. Clinical trial registration: NCT06069687 .

PurposeTo report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector.DesignSingle-center, retrospective chart review.MethodsIn this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated.ResultsThree out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector.ConclusionsWe observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes.Translational relevanceCaution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.

The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology. To demonstrate the feasibility of the developed technology, MAGE is applied to correct the mutated MeCP2 gene in induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) from a Rett syndrome patient. By combining magnetofection and magnetic-activated cell sorting, MAGE achieves higher multi-plasmid delivery (99.3%) and repairing efficiencies (42.95%) with significantly shorter incubation times than conventional transfection agents without size limitations on plasmids. The repaired iPSC-NPCs showed similar characteristics as wild-type neurons when they differentiated into neurons, further validating MAGE and its potential for future clinical applications. In short, the developed nanobio-combined CRISPR-Cas9 technology offers the potential for various clinical applications, particularly in stem cell therapies targeting different genetic diseases.

BACKGROUND: Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM. RESULTS: In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics. CONCLUSIONS: In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN.

During the past 20 years, gene editing has emerged as a novel form of gene therapy. Since the publication of the first potentially therapeutic gene editing platform for genetic disorders, increasingly sophisticated editing technologies have been developed. As with viral vector-mediated gene addition, inborn errors of immunity are excellent candidate diseases for a corrective autologous hematopoietic stem cell gene editing strategy. Research on gene editing for inborn errors of immunity is still entirely preclinical, with no trials yet underway. However, with editing techniques maturing, scientists are investigating this novel form of gene therapy in context of an increasing number of inborn errors of immunity. Here, we present an overview of these studies and the recent progress moving these technologies closer to clinical benefit.

Somatic gene therapy will be one of the most exciting practices of genetic medicine in Africa and is primed to offer a new life for persons living with sickle cell disease (SCD). Recently, successful gene therapy trials for SCD in the USA have sparked a ray of hope within the SCD community in Africa. However, the high cost, estimated to exceed 1.5 million USD, continues to be a major concern for many stakeholders. While affordability is a key global health equity consideration, it is equally important to reflect on other ethical, legal and social issues (ELSIs) that may impact the responsible implementation of gene therapy for SCD in Africa. These include informed consent comprehension, risk of therapeutic misestimation and optimistic bias; priorities for SCD therapy trials; dearth of ethical and regulatory oversight for gene therapy in many African countries; identifying a favourable risk-benefit ratio; criteria for the selection of trial participants; decisional conflict in consent; standards of care; bounded justice; and genetic tourism. Given these ELSIs, we suggest that researchers, pharma, funders, global health agencies, ethics committees, science councils and SCD patient support/advocacy groups should work together to co-develop: (1) patient-centric governance for gene therapy in Africa, (2) public engagement and education materials, and (3) decision making toolkits for trial participants. It is also critical to establish harmonised ethical and regulatory frameworks for gene therapy in Africa, and for global health agencies to accelerate access to basic care for SCD in Africa, while simultaneously strengthening capacity for gene therapy.

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for gene therapies. AAVs have been successfully engineered-for instance, for more efficient and/or cell-specific delivery to numerous tissues-by creating large, diverse starting libraries and selecting for desired properties. However, these starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a prerequisite for any gene delivery goal. Here, we present and showcase a machine learning (ML) method for designing AAV peptide insertion libraries that achieve fivefold higher packaging fitness than the standard NNK library with negligible reduction in diversity. To demonstrate our ML-designed librarys utility for downstream engineering goals, we show that it yields approximately 10-fold more successful variants than the NNK library after selection for infection of human brain tissue, leading to a promising glial-specific variant. Moreover, our design approach can be applied to other types of libraries for AAV and beyond.

Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).

Usher syndrome type 1B (USH1B) is a deaf-blindness disorder, caused by mutations in the MYO7A gene, which encodes the heavy chain of an unconventional actin-based motor protein. Here, we examined the two retinal isoforms of MYO7A, IF1 and IF2. We compared 3D models of the two isoforms and noted that the 38-amino acid region that is present in IF1 but absent from IF2 affects the C lobe of the FERM1 domain and the opening of a cleft in this potentially important protein binding domain. Expression of each of the two isoforms of human MYO7A and pig and mouse Myo7a was detected in the RPE and neural retina. Quantification by qPCR showed that the expression of IF2 was typically ∼ 7-fold greater than that of IF1. We discuss the implications of these findings for any USH1B gene therapy strategy. Given the current incomplete knowledge of the functions of each isoform, both isoforms should be considered for targeting both the RPE and the neural retina in gene augmentation therapies.

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.

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is an inborn error of metabolism caused by inactivating mutations in SGPL1, the gene encoding sphingosine-1-phosphate lyase (SPL), an essential enzyme needed to degrade sphingolipids. SPLIS features include glomerulosclerosis, adrenal insufficiency, neurological defects, ichthyosis, and immune deficiency. Currently, there is no cure for SPLIS, and severely affected patients often die in the first years of life. We reported that adeno-associated virus (AAV) 9-mediated SGPL1 gene therapy (AAV-SPL) given to newborn Sgpl1 knockout mice that model SPLIS and die in the first few weeks of life prolonged their survival to 4.5 months and prevented or delayed the onset of SPLIS phenotypes. In this study, we tested the efficacy of a modified AAV-SPL, which we call AAV-SPL 2.0, in which the original cytomegalovirus (CMV) promoter driving the transgene is replaced with the synthetic CAG promoter used in several clinically approved gene therapy agents. AAV-SPL 2.0 infection of human embryonic kidney (HEK) cells led to 30% higher SPL expression and enzyme activity compared to AAV-SPL. Newborn Sgpl1 knockout mice receiving AAV-SPL 2.0 survived ≥ 5 months and showed normal neurodevelopment, 85% of normal weight gain over the first four months, and delayed onset of proteinuria. Over time, treated mice developed nephrosis and glomerulosclerosis, which likely resulted in their demise. Our overall findings show that AAV-SPL 2.0 performs equal to or better than AAV-SPL. However, improved kidney targeting may be necessary to achieve maximally optimized gene therapy as a potentially lifesaving SPLIS treatment.

Hemophilia is characterized by a deficiency in coagulation factors VIII or IX. The general standard of care for severe hemophilia is frequent intravenous recombinant or plasma-derived factor replacement to prevent bleeding. While this treatment is effective in preventing bleeding, frequent infusions are burdensome for patients. Nonadherence to the therapeutic regimen leaves people with hemophilia at risk for spontaneous and traumatic bleeds into joints as well as life-threatening bleeds such as intracranial hemorrhage. The chronicity of the disorder often leads to the formation of target joints, causing long-term pain and impairing mobility. As a monogenic disorder with well-understood genetics, hemophilia is an ideal disorder for implementing innovations in gene therapies. Indeed, recent approvals of two gene therapy products have the potential to shift the hemophilia treatment paradigm. Valoctocogene roxaparvovec and etranacogene dezaparvovec-drlb are gene therapies for hemophilia A and B, respectively. These therapies, given as a single intravenous infusion, may improve patients quality of life, decreasing treatment burden and resulting in factor expression that virtually eliminates the need for factor replacement. Since both treatments involve viral vectors targeted to the liver, short- and long-term safety and efficacy monitoring involves monitoring liver enzymes to track liver health. Long-term monitoring of efficacy, durability of gene expression, and safety are ongoing. Gene therapy presents a promising new therapeutic option for patients with hemophilia and warrants continued innovation and investigation.

Genome-editing technologies have ushered in a new era in gene therapy, providing novel therapeutic strategies for a wide range of diseases, including both genetic and nongenetic ocular diseases. These technologies offer new hope for patients suffering from previously untreatable conditions. The unique anatomical and physiological features of the eye, including its immune-privileged status, size, and compartmentalized structure, provide an optimal environment for the application of these cutting-edge technologies. Moreover, the development of various delivery methods has facilitated the efficient and targeted administration of genome engineering tools designed to correct specific ocular tissues. Additionally, advancements in noninvasive ocular imaging techniques and electroretinography have enabled real-time monitoring of therapeutic efficacy and safety. Herein, we discuss the discovery and development of genome-editing technologies, their application to ocular diseases from the anterior segment to the posterior segment, current limitations encountered in translating these technologies into clinical practice, and ongoing research endeavors aimed at overcoming these challenges.

MicroRNA-22 (miR-22) can be induced by beneficial metabolites that have metabolic and immune effects, including retinoic acids, bile acids, vitamin D3, and short-chain fatty acids. The tumor suppressor effects of miR-22 have been suggested, but whether miR-22 treats orthotopic hepatocellular carcinoma (HCC) is not established. The role of miR-22 in regulating tumor immunity is also poorly understood. Our data showed that miR-22 delivered by adeno-associated virus serotype 8 effectively treated HCC. Compared with FDA-approved lenvatinib, miR-22 produced better survival outcomes without noticeable toxicity. miR-22 silenced hypoxia-inducible factor 1 (HIF1α) and enhanced retinoic acid signaling in both hepatocytes and T cells. Moreover, miR-22 treatment improved metabolism and reduced inflammation. In the liver, miR-22 reduced the abundance of IL17-producing T cells and inhibited IL17 signaling by reducing the occupancy of HIF1α in the Rorc and Il17a genes. Conversely, increasing IL17 signaling ameliorated the anti-HCC effect of miR-22. Additionally, miR-22 expanded cytotoxic T cells and reduced regulatory T cells (Treg). Moreover, depleting cytotoxic T cells also abolished the anti-HCC effects of miR-22. In patients, miR-22 high HCC had upregulated metabolic pathways and reduced IL17 pro-inflammatory signaling compared with miR-22 low HCC. Together, miR-22 gene therapy can be a novel option for HCC treatment.

DISCLOSURES: Dr Tice and Mr Sarker received ICER grants during the conduct of the study. Dr Moradi, Ms Herce-Hagiwara, Dr Faghim, Dr Agboola, Dr Rind, and Dr Pearson reports grants from Arnold Ventures, grants from Blue Cross Blue Shield of MA, grants from California Healthcare Foundation, grants from The Commonwealth Fund, grants from The Peterson Center on Healthcare, during the conduct of the study; other from Aetna, other from Americas Health Insurance Plans, other from Anthem, other from AbbVie, other from Alnylam, other from AstraZeneca, other from Biogen, other from Blue Shield of CA, other from Cambia Health Services, other from CVS, other from Editas, other from Express Scripts, other from Genentech/Roche, other from GlaxoSmithKline, other from Harvard Pilgrim, other from Health Care Service Corporation, other from Health Partners, other from Johnson & Johnson (Janssen), other from Kaiser Permanente, other from LEO Pharma, other from Mallinckrodt, other from Merck, other from Novartis, other from National Pharmaceutical Council, other from Premera, other from Prime Therapeutics, other from Regeneron, other from Sanofi, other from Spark Therapeutics, other from United Healthcare, other from HealthFirst, other from Pfizer, other from Boehringer-Ingelheim, other from uniQure, other from Evolve Pharmacy Solutions, other from Humana, other from Sun Life, outside the submitted work.

Gene augmentation and genome editing are promising strategies for the treatment of monogenic inherited retinal diseases. Although gene augmentation treatments are commercially available for inherited retinal diseases, there are many shortcomings that need to be addressed, like progressive retinal degeneration and diminishing efficacy over time. Innovative CRISPR-Cas9-based genome editing technologies have broadened the proportion of treatable genetic disorders and can greatly improve or complement treatment outcomes from gene augmentation. Progress in this relatively new field involves the development of therapeutics including gene disruption, ablate-and-replace strategies, and precision gene correction techniques, such as base editing and prime editing. By making direct edits to endogenous DNA, genome editing theoretically guarantees permanent gene correction and long-lasting treatment effects. Improvements to delivery modalities aimed at limiting persistent gene editor activity have displayed an improved safety profile and minimal off-target editing. Continued progress to advance precise gene correction and associated delivery strategies will establish genome editing as the preferred treatment for genetic retinal disorders. This commentary describes the applications, strengths, and drawbacks of conventional gene augmentation approaches, recent advances in precise genome editing in the retina, and promising preclinical strategies to facilitate the use of robust genome editing therapies in human patients.

Duchenne muscular dystrophy (DMD) is a serious, rare genetic disease, affecting primarily boys. It is caused by mutations in the DMD gene and is characterized by progressive muscle degeneration that results in loss of function and early death due to respiratory and/or cardiac failure. Although limited treatment options are available, some for only small subsets of the patient population, DMD remains a disease with large unmet medical needs. The adeno-associated virus (AAV) vector is the leading gene delivery system for addressing genetic neuromuscular diseases. Since the gene encoding the full-length dystrophin protein exceeds the packaging capacity of a single AAV vector, gene replacement therapy based on AAV-delivery of shortened, yet, functional microdystrophin genes has emerged as a promising treatment. This article seeks to explain the rationale for use of the accelerated approval pathway to advance AAV microdystrophin gene therapy for DMD. Specifically, we provide support for the use of microdystrophin expression as a surrogate endpoint that could be used in clinical trials to support accelerated approval.

Leber hereditary optic neuropathy (LHON) is an important example of mitochondrial blindness with the m.11778G>A mutation in the MT-ND4 gene being the most common disease-causing mtDNA variant worldwide. The REFLECT phase 3 pivotal study is a randomized, double-masked, placebo-controlled trial investigating the efficacy and safety of bilateral intravitreal injection of lenadogene nolparvovec in patients with a confirmed m.11778G>A mutation, using a recombinant adeno-associated virus vector 2, serotype 2 (rAAV2/2-ND4). The first-affected eye received gene therapy; the fellow (affected/not-yet-affected) eye was randomly injected with gene therapy or placebo. The primary end point was the difference in change from baseline of best-corrected visual acuity (BCVA) in second-affected/not-yet-affected eyes treated with lenadogene nolparvovec versus placebo at 1.5 years post-treatment, expressed in logarithm of the minimal angle of resolution (LogMAR). Forty-eight patients were treated bilaterally and 50 unilaterally. At 1.5 years, the change from baseline in BCVA was not statistically different between second-affected/not-yet-affected eyes receiving lenadogene nolparvovec and placebo (primary end point). A statistically significant improvement in BCVA was reported from baseline to 1.5 years in lenadogene nolparvovec-treated eyes: -0.23 LogMAR for the first-affected eyes of bilaterally treated patients (P < 0.01); and -0.15 LogMAR for second-affected/not-yet-affected eyes of bilaterally treated patients and the first-affected eyes of unilaterally treated patients (P < 0.05). The mean improvement in BCVA from nadir to 1.5 years was -0.38 (0.052) LogMAR and -0.33 (0.052) LogMAR in first-affected and second-affected/not-yet-affected eyes treated with lenadogene nolparvovec, respectively (bilateral treatment group). A mean improvement of -0.33 (0.051) LogMAR and -0.26 (0.051) LogMAR was observed in first-affected lenadogene nolparvovec-treated eyes and second-affected/not-yet-affected placebo-treated eyes, respectively (unilateral treatment group). The proportion of patients with one or both eyes on-chart at 1.5 years was 85.4% and 72.0% for bilaterally and unilaterally treated patients, respectively. The gene therapy was well tolerated, with no systemic issues. Intraocular inflammation, which was mostly mild and well controlled with topical corticosteroids, occurred in 70.7% of lenadogene nolparvovec-treated eyes versus 10.2% of placebo-treated eyes. Among eyes treated with lenadogene nolparvovec, there was no difference in the incidence of intraocular inflammation between bilaterally and unilaterally treated patients. Overall, the REFLECT trial demonstrated an improvement of BCVA in LHON eyes carrying the m.11778G>A mtDNA mutation treated with lenadogene nolparvovec or placebo to a degree not reported in natural history studies and supports an improved benefit/risk profile for bilateral injections of lenadogene nolparvovec relative to unilateral injections.

The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.

Cystinosis is an autosomal recessive metabolic disease characterized by lysosomal accumulation of cystine in all the cells of the body. Infantile cystinosis begins in infancy by a renal Fanconi syndrome and eventually leads to multi-organ failure, including the kidney, eye, thyroid, muscle, and pancreas, eventually causing premature death in early adulthood. The current treatment is the drug cysteamine that only delays the progression of the disease. We identified the gene involved, CTNS, and showed that the encoded protein, cystinosin, is a proton-driven cystine transporter. We generated a mouse model of cystinosis, the Ctns-/- mice, that recapitulates the main disease complications. The goal was next to develop a gene therapy approach for cystinosis. We used bone marrow stem cells as a vehicle to bring the healthy CTNS gene to tissues, and we showed that wild-type hematopoietic stem and progenitor cell (HSPC) transplantation led to abundant tissue integration of bone marrow-derived cells, significant decrease of tissue cystine accumulation and long-term kidney, eye and thyroid preservation. We then developed an autologous transplantation approach of HSPCs modified ex vivo using a lentiviral vector to introduce a functional CTNS cDNA, and showed its efficacy in Ctns-/- mice. We conducted the pharmacology/toxicology studies, developed the manufacturing process using human CD34+ cells, and design the clinical trial. We received Food and Drug Administration (FDA)-clearance to start a phase 1/2 clinical trial for cystinosis in December 2018. Six patients have been treated so far. In this review, we describe the path to go from the gene to a gene therapy approach for cystinosis.

Sickle cell disease (SCD) results from a single base pair change in the sixth codon of the β-globin chain of hemoglobin, which promotes aggregation of deoxyhemoglobin, increasing rigidity of red blood cells and causing vaso-occlusive and hemolytic complications. Allogeneic transplant of hematopoietic stem cells (HSCs) can eliminate SCD manifestations but is limited by absence of well-matched donors and immune complications. Gene therapy with transplantation of autologous HSCs that are gene-modified may provide similar benefits without the immune complications. Much progress has been made, and patients are realizing significant clinical improvements in multiple trials using different approaches with lentiviral vector-mediated gene addition to inhibit hemoglobin aggregation. Gene editing approaches are under development to provide additional therapeutic opportunities. Gene therapy for SCD has advanced from an attractive concept to clinical reality.

With the advent of recombinant DNA technology in the 1970s, the idea of using gene therapies to treat human genetic diseases captured the interest and imagination of scientists around the world. Years later, enabled largely by the development of CRISPR-based genome editing tools, the field has exploded, with academic labs, startup biotechnology companies, and large pharmaceutical corporations working in concert to develop life-changing therapeutics. In this Essay, we highlight base editing technologies and their development from bench to bedside. Base editing, first reported in 2016, is capable of installing C•G to T•A and A•T to G•C point mutations, while largely circumventing some of the pitfalls of traditional CRISPR/Cas9 gene editing. Despite their youth, these technologies have been widely used by both academic labs and therapeutics-based companies. Here, we provide an overview of the mechanics of base editing and its use in clinical trials.

Investigations to understand the function and control of the globin genes have led to some of the most exciting molecular discoveries and biomedical breakthroughs of the 20th and 21st centuries. Extensive characterization of the globin gene locus, accompanied by pioneering work on the utilization of viruses as human gene delivery tools in human hematopoietic stem and progenitor cells (HPSCs), has led to transformative and successful therapies via autologous hematopoietic stem-cell transplant with gene therapy (HSCT-GT). Due to the advanced understanding of the β-globin gene cluster, the first diseases considered for autologous HSCT-GT were two prevalent β-hemoglobinopathies: sickle cell disease and β-thalassemia, both affecting functional β-globin chains and leading to substantial morbidity. Both conditions are suitable for allogeneic HSCT; however, this therapy comes with serious risks and is most effective using an HLA-matched family donor (which is not available for most patients) to obtain optimal therapeutic and safe benefits. Transplants from unrelated or haplo-identical donors carry higher risks, although they are progressively improving. Conversely, HSCT-GT utilizes the patient's own HSPCs, broadening access to more patients. Several gene therapy clinical trials have been reported to have achieved significant disease improvement, and more are underway. Based on the safety and the therapeutic success of autologous HSCT-GT, the U.S. Food and Drug Administration (FDA) in 2022 approved an HSCT-GT for β-thalassemia (Zynteglo™). This review illuminates the β-globin gene research journey, adversities faced, and achievements reached; it highlights important molecular and genetic findings of the β-globin locus, describes the predominant globin vectors, and concludes by describing promising results from clinical trials for both sickle cell disease and β-thalassemia.

lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) comprises autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified β-globin gene (βA-T87Q ) to produce anti-sickling hemoglobin (HbAT87Q ). The efficacy and safety of lovo-cel for SCD are being evaluated in the ongoing phase 1/2 HGB-206 study (ClinicalTrials.gov: NCT02140554). The treatment process evolved over time, using learnings from outcomes in the initial patients to optimize lovo-cel's benefit-risk profile. Following modest expression of HbAT87Q in the initial patients (Group A, n = 7), alterations were made to the treatment process for patients subsequently enrolled in Group B (n = 2, patients B1 and B2), including improvements to cell collection and lovo-cel manufacturing. After 6 months, median Group A peripheral blood vector copy number (≥0.08 c/dg) and HbAT87Q levels (≥0.46 g/dL) were inadequate for substantial clinical effect but stable and sustained over 5.5 years; both markedly improved in Group B (patient B1: ≥0.53 c/dg and ≥2.69 g/dL; patient B2: ≥2.14 c/dg and ≥6.40 g/dL, respectively) and generated improved biologic and clinical efficacy in Group B, including higher total hemoglobin and decreased hemolysis. The safety of the lovo-cel for SCD treatment regimen largely reflected the known side effects of HSPC collection, busulfan conditioning regimen, and underlying SCD; acute myeloid leukemia was observed in two patients in Group A and deemed unlikely related to insertional oncogenesis. Changes made during development of the lovo-cel treatment process were associated with improved outcomes and provide lessons for future SCD GT studies.

BackgroundThe DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation.MethodsWe carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months.ResultsMarrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor β chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report.ConclusionsInfusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

Curative therapies for sickle cell disease (SCD) include allogeneic human leukocyte antigen (HLA)- matched sibling and haploidentical hematopoietic cell transplant (HCT), gene therapy, and gene editing. However, comparative trial data that might facilitate selecting one curative therapy over another are unavailable. New strategies to decrease graft rejection and graft-versus-host disease (GVHD) risks are needed to expand haploidentical HCT. Myeloablative gene therapy and gene editing also has limitations. Herein, we review recent studies on curative therapies for SCD in the past 5 years.

Adeno-associated viruses (AAVs) are being increasingly used as gene therapy vectors in clinical studies especially targeting central nervous system (CNS) disorders. Correspondingly, host immune responses to the AAV capsid or the transgene-encoded protein have been observed in various clinical and preclinical studies. Such immune responses may adversely impact patients' health, prevent viral transduction, prevent repeated dosing strategies, eliminate transduced cells, and pose a significant barrier to the potential effectiveness of AAV gene therapy. Consequently, multiple immunomodulatory strategies have been used in attempts to limit immune-mediated responses to the vector, enable readministration of AAV gene therapy, prevent end-organ toxicity, and increase the duration of transgene-encoded protein expression. Herein we review the innate and adaptive immune responses that may occur during CNS-targeted AAV gene therapy as well as host- and treatment-specific factors that could impact the immune response. We also summarize the available preclinical and clinical data on immune responses specifically to CNS-targeted AAV gene therapy and discuss potential strategies for incorporating prophylactic immunosuppression regimens to circumvent adverse immune responses.

Autologous stem cell transplant with gene therapy (ASCT-GT) provides curative therapy while reducing pretransplant immune-suppressive conditioning and eliminating posttransplant immune suppression. Clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations increase and telomere lengths (TLs) shorten with natural aging and DNA damaging processes. It is possible that, if CHIP is present before ASCT-GT or mutagenesis occurs after busulfan exposure, the hematopoietic stem cells carrying these somatic variants may survive the conditioning chemotherapy and have a selective reconstitution advantage, increasing the risk of hematologic malignancy and overall mortality. Seventy-four peripheral blood samples (ranging from baseline to 120 months after ASCT-GT) from 10 pediatric participants who underwent ASCT-GT for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) after reduced-intensity conditioning with busulfan and 16 healthy controls were analyzed for TL and CHIP. One participant had a significant decrease in TL. There were no CHIP-associated mutations identified by the next-generation sequencing in any of the ADA-SCID participants. This suggests that further studies are needed to determine the utility of germline analyses in revealing the underlying genetic risk of malignancy in participants who undergo gene therapy. Although these results are promising, larger scale studies are needed to corroborate the effect of ASCT-GT on TL and CHIP. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Inherited retinal degenerations such as retinitis pigmentosa (RP) affect around one in 4000 people and are the leading cause of blindness in working age adults in several countries. In these typically monogenic conditions, there is progressive degeneration of photoreceptors; however, inner retinal neurons such as bipolar cells and ganglion cells remain largely structurally intact, even in end-stage disease. Therapeutic approaches aiming to stimulate these residual cells, independent of the underlying genetic cause, could potentially restore visual function in patients with advanced vision loss, and benefit many more patients than therapies directed at the specific gene implicated in each disorder. One approach investigated for this purpose is that of optogenetics, a method of neuromodulation that utilises light to activate neurons engineered to ectopically express a light-sensitive protein. Using gene therapy via adeno-associated viral vectors, a range of photosensitive proteins have been expressed in remaining retinal cells in advanced retinal degeneration with in vivo studies demonstrating restoration of visual function. Developing an effective optogenetic strategy requires consideration of multiple factors, including the light-sensitive protein that is used, the vector and method for gene delivery, and the target cell for expression because these in turn may affect the quality of vision that can be restored. Currently, at least four clinical trials are ongoing to investigate optogenetic therapies in patients, with the ultimate aim of reversing visual loss in end-stage disease.

We are living in a golden age of medicine in which the availability of prenatal diagnosis, fetal therapy, and gene therapy/editing make it theoretically possible to repair almost any defect in the genetic code. Furthermore, the ability to diagnose genetic disorders before birth and the presence of established surgical techniques enable these therapies to be delivered safely to the fetus. Prenatal therapies are generally used in the second or early third trimester for severe, life-threatening disorders for which there is a clear rationale for intervening before birth. While there has been promising work for prenatal gene therapy in preclinical models, the path to a clinical prenatal gene therapy approach is complex. We recently held a conference with the University of California, San Francisco-Stanford Center of Excellence in Regulatory Science and Innovation, researchers, patient advocates, regulatory (members of the Food and Drug Administration), and other stakeholders to review the scientific background and rationale for prenatal somatic cell gene therapy for severe monogenic diseases and initiate a dialogue toward a safe regulatory path for phase 1 clinical trials. This review represents a summary of the considerations and discussions from these conversations.

Granulocyte transfusions are sometimes used as adjunctive therapy for the treatment of infection in patients with chronic granulomatous disease (CGD). However, granulocyte transfusions can be associated with a high rate of alloimmunization, and their role in CGD patients undergoing hematopoietic cell transplantation (HCT) or gene therapy (GT) is unknown. We identified 27 patients with CGD who received granulocyte transfusions pre- (within 6 months) and/or post-HCT or GT in a retrospective survey. Twelve patients received granulocyte transfusions as a bridge to cellular therapy. Six (50%) of these patients had a complete or partial response. However, six of 10 (60%) patients for whom testing was performed developed anti-HLA antibodies, and three of the patients also had severe immune-mediated cytopenia within the first 100 days post-HCT or GT. Fifteen patients received granulocyte transfusions post-HCT only. HLA antibodies were not checked for any of these 15 patients, but there were no cases of early immune-mediated cytopenia. Out of 25 patients who underwent HCT, there were 5 (20%) cases of primary graft failure. Three of the patients with primary graft failure had received granulocyte transfusions pre-HCT and were subsequently found to have anti-HLA antibodies. In this small cohort of patients with CGD, granulocyte transfusions pre-HCT or GT were associated with high rates of alloimmunization, primary graft failure, and early severe immune-mediated cytopenia post-HCT or GT. Granulocyte transfusions post-HCT do not appear to confer an increased risk of graft failure.

BackgroundThe regenerative capacity of the heart after myocardial infarction is limited. Our previous study showed that ectopic introduction of 4 cell cycle factors (4F; CDK1 [cyclin-dependent kinase 1], CDK4 [cyclin-dependent kinase 4], CCNB [cyclin B1], and CCND [cyclin D1]) promotes cardiomyocyte proliferation in 15% to 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction in mice.MethodsUsing temporal single-cell RNA sequencing, we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Using rat and pig models of ischemic heart failure, we aimed to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure.ResultsTemporal bulk and single-cell RNA sequencing and further biochemical validations of mature human induced pluripotent stem cell-derived cardiomyocytes treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 hours after infection with 4F, which was associated mainly with sarcomere disassembly and metabolic reprogramming (n=3/time point/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic nonintegrating lentivirus (NIL) encoding 4F; each is driven by a TNNT2 (cardiac troponin T isoform 2) promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially 1 week after myocardial infarction in rats (n=10/group) or pigs (n=6-7/group). Four weeks after injection, TNNT2-4Fpolycistronic-NIL-treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus-treated animals. At 4 months after treatment, rats that received TNNT2-4Fpolycistronic-NIL still showed a sustained improvement in cardiac function and no obvious development of cardiac arrhythmias or systemic tumorigenesis (n=10/group).ConclusionsThis study provides mechanistic insights into the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors owing to the use of a novel transient and cardiomyocyte-specific viral construct.

This paper explores the potential of rAAV2-retro to deliver gene modifying cargoes to the cells of origin of multiple pathways that are interrupted by spinal cord injury (SCI), summarizing data from previous studies and new data from additional experiments. rAAV-retro exhibits uniquely robust and reliable long-distance retrograde transport from pre-terminal axons and synapses back to neuronal bodies. Previous studies have documented that various AAV-based genetic modifications can enable axon regeneration after SCI, but these have targeted the cells of origin of one pathway at a time. In contrast, rAAV-retro can simultaneously transduce large numbers of neurons of origin of multiple spinal pathways with single injections into the spinal cord. Our initial studies use RosatdTomato and double transgenic PTENf/f; RosatdTomato mice in which transfection with rAAV-retro/Cre deletes PTEN and activates tdT expression in the same neurons. Injections of rAAV-retro/Cre into the cervical, thoracic and lumbar spinal cord led to topographically specific retrograde transduction in cortical motoneurons and neurons in subcortical regions that give rise to different spinal pathways. Our results confirm and extend previous studies indicating selective transduction of neurons that terminate at the level of the injection with minimal retrograde transduction of axons in transit to lower levels. We document feasibility of using rAAV-retro expressing shRNA against PTEN along with a GFP reporter (rAAV-retro-shPTEN/GFP) to effectively knock down PTEN in multiple populations of neurons, which can be used in any species. Some limitations and caveats of currently available rAAV-retros are discussed. Together, our results support the potential applications of rAAV-retro for AAV-based gene-modifications for SCI.

Gene therapies aim to deliver a therapeutic payload to specified tissues with underlying protein deficiency. Since the 1990s, gene therapies have been explored as potential treatments for chronic conditions requiring lifetime care and medical management. Ocular gene therapies target a range of ocular disorders, but retinal diseases are of particular importance due to the prevalence of retinal disease and the current treatment burden of such diseases on affected patients, as well as the challenge of properly delivering these therapies to the target tissue. The purpose of this review is to provide an update on the most current data available for five different retinal gene therapies currently undergoing clinical trials for use against age-related macular degeneration (AMD) and the development of novel delivery routes for the administration of such therapies. Research has been performed and compiled from PubMed and the select authors of this manuscript on the treatment and effectiveness of five current retinal gene therapies: Luxturna, ADVM-022, RGX-314, GT-005, and HMR59. We present the available data of current clinical trials for the treatment of neovascular and dry age-related macular degeneration with different AAV-based gene therapies. We also present current research on the progress of developing novel routes of administration for ocular gene therapies. Retinal gene therapies offer the potential for life-changing treatment for chronic conditions like age-related macular degeneration with a single administration. In doing so, gene therapies change the landscape of treatment options for these chronic conditions for both patient and provider.

Background and objectivesTo report final, 36-month safety and clinical outcomes from the PD-1101 trial of NBIb-1817 (VY-AADC01) in participants with moderately advanced Parkinson disease (PD) and motor fluctuations.MethodsPD-1101 was a phase 1b, open-label, dose escalation trial of VY-AADC01, an experimental AAV2 gene therapy encoding the human aromatic l-amino acid decarboxylase (AADC) enzyme. VY-AADC01 was delivered via bilateral, intraoperative MRI-guided putaminal infusions to 3 cohorts (n = 5 participants per cohort): cohort 1, ≤7.5 × 1011 vector genomes (vg); cohort 2, ≤1.5 × 1012 vg; cohort 3, ≤4.7 × 1012 vg.ResultsNo serious adverse events (SAEs) attributed to VY-AADC01 were reported. All 4 non-vector-related SAEs (atrial fibrillation and pulmonary embolism in 1 participant and 2 events of small bowel obstruction in another participant) resolved. Requirements for PD medications were reduced by 21%-30% in the 2 highest dose cohorts at 36 months. Standard measures of motor function (PD diary, Unified Parkinson's Disease Rating Scale III "off"-medication and "on"-medication scores), global impressions of improvement (Clinical Global Impression of Improvement, Patient Global Impression of Improvement), and quality of life (39-item Parkinson's Disease Questionnaire) were stable or improved compared with baseline at 12, 24, and 36 months following VY-AADC01 administration across cohorts.DiscussionsVY-AADC01 and the surgical administration procedure were well-tolerated and resulted in stable or improved motor function and quality of life across cohorts, as well as reduced PD medication requirements in cohorts 2 and 3 over 3 years.Trial registration informationNCT01973543.Classification of evidenceThis study provides Class IV evidence that, in patients with moderately advanced PD and motor fluctuations, putaminal infusion of VY-AADC01 is well tolerated and may improve motor function.

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety.

BACKGROUND: Gene-targeted therapies are becoming a reality for infants and children with diseases of the nervous system. Rapid scientific advances have led to disease-modifying or even curative treatments. However, delays and gaps in diagnosis, inequitable delivery, and the need for long-term surveillance pose unresolved challenges. OBJECTIVE AND METHODS: The goal of the Child Neurology Society Research Committee was to evaluate and provide guidance on the obstacles, opportunities, and uncertainties in gene-targeted therapies for pediatric neurological disease. The Child Neurology Society Research Committee engaged in collaborative, iterative literature review and committee deliberations to prepare this consensus statement. RESULTS: We identified important challenges for gene-targeted therapies that require resource investments, infrastructure development, and strategic planning. Barriers include inequities in diagnosis and delivery of therapies, high costs, and a need for long-term surveillance of efficacy and safety, including systematic tracking of unanticipated effects. Key uncertainties regarding technical aspects and usage of gene-targeted therapies should be addressed, and characterization of new natural histories of diseases will be needed. Counterbalanced with these obstacles and uncertainties is the tremendous potential being demonstrated in treatments and clinical trials of gene-targeted therapies. CONCLUSIONS: Given that gene-targeted therapies for neurological diseases are in their earliest phase, the pediatric neurology community can play a vital role in their guidance and implementation. This role includes facilitating development of infrastructure and guidelines; ensuring efficient, equitable, and ethical implementation of treatments; and advocating for affordable and broad access for all children.

Regional gene therapy using a lentiviral vector containing the BMP-2 complementary DNA (cDNA) has been shown to heal critical-sized bone defects in rodent models. An appropriate "cellular dose" needs to be defined for eventual translation into human trials. The purpose of this study was to evaluate bone defect healing potential and quality using three different doses of transduced human bone marrow cells (HBMCs). HBMCs were transduced with a lentiviral vector containing either BMP-2 or green fluorescent protein (GFP). All cells were loaded onto compression-resistant matrices and implanted in the bone defect of athymic rats. Treatment groups included femoral defects that were treated with a low-dose (1 × 106 cells), standard-dose (5 × 106 cells), and high-dose (1.5 × 107 cells) HBMCs transduced with lentiviral vector containing BMP-2 cDNA. The three control groups were bone defects treated with HBMCs that were either nontransduced or transduced with vector containing GFP. All animals were sacrificed at 12 weeks. The bone formed in each defect was evaluated with plain radiographs, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. Bone defects treated with higher doses of BMP-2-producing cells were more likely to have healed (6/14 of the low-dose group; 12/14 of the standard-dose group; 14/14 of the high-dose group; χ2(2) = 15.501, p

The development of CRISPR-derived genome editing technologies has enabled the precise manipulation of DNA sequences within the human genome. In this review, we discuss the initial development and cellular mechanism of action of CRISPR nucleases and DNA base editors. We then describe factors that must be taken into consideration when developing these tools into therapeutic agents, including the potential for unintended and off-target edits when using these genome editing tools, and methods to characterize these types of edits. We finish by considering specific challenges associated with bringing a CRISPR-based therapy to the clinic, including manufacturing, regulatory oversight, and considerations for clinical trials that involve genome editing agents.

PurposeAdenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) through an accumulation of toxic metabolites within lymphocytes. Recently, ADA deficiency has been successfully treated using lentiviral-transduced autologous CD34+ cells carrying the ADA gene. T and B cell function appears to be fully restored, but in many patients' B cell numbers remain low, and assessments of the immunoglobulin heavy (IgHV) repertoire following gene therapy are lacking.MethodsWe performed deep sequencing of IgHV repertoire in peripheral blood lymphocytes from a child following lentivirus-based gene therapy for ADA deficiency and compared to the IgHV repertoire in healthy infants and adults.ResultsAfter gene therapy, Ig diversity increased over time as evidenced by V, D, and J gene usage, N-additions, CDR3 length, extent of somatic hypermutation, and Ig class switching. There was the emergence of predominant IgHM, IgHG, and IgHA CDR3 lengths after gene therapy indicating successful oligoclonal expansion in response to antigens. This provides proof of concept for the feasibility and utility of molecular monitoring in following B cell reconstitution following gene therapy for ADA deficiency.ConclusionBased on deep sequencing, gene therapy resulted in an IgHV repertoire with molecular diversity similar to healthy infants.

OBJECTIVE: This report presents a cross-sectional analysis of the baseline characteristics of subjects with Leber hereditary optic neuropathy enrolled in the gene therapy trials RESCUE and REVERSE, to illustrate the evolution of visual parameters over the first year after vision loss. METHODS: RESCUE and REVERSE were 2 phase III clinical trials designed to assess the efficacy of rAAV2/2-ND4 gene therapy in ND4-LHON subjects. At enrollment, subjects had vision loss for ≤6 months in RESCUE, and between 6 and 12 months in REVERSE. Functional visual parameters (best-corrected visual acuity [BCVA], contrast sensitivity [CS], and Humphrey Visual Field [HVF]) and structural parameters assessed by spectral-domain optical coherence tomography were analyzed in both cohorts before treatment. The cross-sectional analysis of functional and anatomic parameters included the baseline values collected in all eyes at 2 different visits (Screening and Inclusion). RESULTS: Seventy-six subjects were included in total, 39 in RESCUE and 37 in REVERSE. Mean BCVA was significantly worse in RESCUE subjects compared with REVERSE subjects (1.29 and 1.61 LogMAR respectively, P = 0.0029). Similarly, mean CS and HVF were significantly more impaired in REVERSE vs RESCUE subjects (P < 0.005). The cross-sectional analysis showed that the monthly decrease in BCVA, ganglion cell layer macular volume, and retinal nerve fiber layer thickness was much more pronounced in the first 6 months after onset (+0.24 LogMAR, -0.06 mm3, and -6.00 μm respectively) than between 6 and 12 months after onset (+0.02 LogMAR, -0.01 mm3, and -0.43 μm respectively). CONCLUSION: LHON progresses rapidly in the first months following onset during the subacute phase, followed by relative stabilization during the dynamic phase.

BACKGROUND: RESCUE and REVERSE were 2 Phase 3 clinical trials that assessed the efficacy and safety of intravitreal gene therapy with lenadogene nolparvovec (rAAV2/2-ND4) for the treatment of Leber hereditary optic neuropathy (LHON). RESTORE is the long-term follow-up study of subjects treated in the RESCUE and REVERSE trials. METHODS: In RESCUE and REVERSE, 76 subjects with LHON because of the m.11778 G>A mutation in the mitochondrial gene ND4 received a single unilateral intravitreal injection of lenadogene nolparvovec. After 96 weeks, 61 subjects were enrolled in the long-term follow-up study RESTORE. The best-corrected visual acuity (BCVA) was assessed over a period of up to 52 months after onset of vision loss. A locally estimated scatterplot smoothing regression model was used to analyze changes in BCVA over time. Vision-related quality of life was reported using the visual function questionnaire-25 (VFQ-25). RESULTS: The population of MT-ND4 subjects enrolled in RESTORE was representative of the combined cohorts of RESCUE and REVERSE for mean age (35.1 years) and gender distribution (79% males). There was a progressive and sustained improvement of BCVA up to 52 months after the onset of vision loss. The final mean BCVA was 1.26 logarithm of the minimal angle of resolution 48 months after the onset of vision loss. The mean VFQ-25 composite score increased by 7 points compared with baseline. CONCLUSION: The treatment effect of lenadogene nolparvovec on BCVA and vision-related quality of life observed 96 weeks (2 years) after treatment in RESCUE and REVERSE was sustained at 3 years in RESTORE, with a maximum follow-up of 52 months (4.3 years) after the onset of vision loss.

Virotherapy, a strategy to use live viruses as therapeutics, is a relatively novel field in the treatment of cancer. With the advancements in molecular biology and virology, there has been a huge increase in research on cancer virotherapy. For the treatment of cancer, viruses could be used either as vectors in gene therapy or as oncolytic agents. A variety of viruses have been studied for their potential usage in gene therapy or oncolytic therapy. In this review, we discuss virotherapy with a special focus on breast cancer. Breast cancer is the most common cancer and the leading cause of cancer-related deaths in women worldwide. Current treatments are insufficient to cure metastatic breast cancer and are often associated with severe side effects that further deteriorates patients' quality of life. Therefore, novel therapeutic approaches such as virotherapy need to be developed for the treatment of breast cancer. Here we summarize the current treatments for breast cancer and the potential use of virotherapy in the treatment of the disease. Furthermore, we discuss the use of oncolytic viruses as immunotherapeutics and the rational combination of oncolytic viruses with other therapeutics for optimal treatment of breast cancer. Finally, we outline the progress made in virotherapy for breast cancer and the shortcomings that need to be addressed for this novel therapy to move to the clinic for better treatment of breast cancer.

IntroductionInfants with SCID are treated with hematopoietic cell transplantation (HCT) or gene therapy (GT). Caregiver perceptions of pre-treatment counseling and understanding of durability of HCT/GT are poorly understood.MethodsA survey was designed and distributed to families of patients with SCID. Topics in the questionnaire included SCID genotype and treatment, family recollections of pre-treatment counseling and present clinical status.Results151 surveys were analyzed. 132 were treated with HCT, 19 with GT. From counseling received, 37% expected HCT/GT would lead to "cure"; 43% expected HCT/GT would last a lifetime. Of 136 living patients, 59% reported overall good health but 65% reported some persistent health challenges.ConclusionsFor some, interpretation of the word "cure" varied, leading to misunderstanding regarding need for continued medical evaluations and additional therapies. Clear communication regarding the importance of lifelong follow-up, no matter the treatment outcome, will help to optimize good health and quality of life.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare genetic disorder characterized by deficient synthesis of dopamine and serotonin. It presents in early infancy, and causes severe developmental disability and lifelong motor, behavioral, and autonomic symptoms including oculogyric crises (OGC), sleep disorder, and mood disturbance. We investigated the safety and efficacy of delivery of a viral vector expressing AADC (AAV2-hAADC) to the midbrain in children with AADC deficiency (ClinicalTrials.gov Identifier NCT02852213). Seven (7) children, aged 4-9 years underwent convection-enhanced delivery (CED) of AAV2-hAADC to the bilateral substantia nigra (SN) and ventral tegmental area (VTA) (total infusion volume: 80 µL per hemisphere) in 2 dose cohorts: 1.3 × 1011 vg (n = 3), and 4.2 × 1011 vg (n = 4). Primary aims were to demonstrate the safety of the procedure and document biomarker evidence of restoration of brain AADC activity. Secondary aims were to assess clinical improvement in symptoms and motor function. Direct bilateral infusion of AAV2-hAADC was safe, well-tolerated and achieved target coverage of 98% and 70% of the SN and VTA, respectively. Dopamine metabolism was increased in all subjects and FDOPA uptake was enhanced within the midbrain and the striatum. OGC resolved completely in 6 of 7 subjects by Month 3 post-surgery. Twelve (12) months after surgery, 6/7 subjects gained normal head control and 4/7 could sit independently. At 18 months, 2 subjects could walk with 2-hand support. Both the primary and secondary endpoints of the study were met. Midbrain gene delivery in children with AADC deficiency is feasible and safe, and leads to clinical improvements in symptoms and motor function.

American Society of Hematology conducts an annual meeting, where investigators from around the globe presented ground-breaking research in the fields of malignant and non-malignant hematology. We provide a summary of non-malignant hematology abstracts from the 2020 meeting. Topics included range from those related to thrombosis, including thrombotic complications of COVID-19, bleeding and novel therapies such as gene therapies. Readers are encouraged to access meeting materials for a more detailed coverage of the event.

Angelman syndrome (AS) is a rare (~1:15,000) neurodevelopmental disorder characterized by severe developmental delay and intellectual disability, impaired communication skills, and a high prevalence of seizures, sleep disturbances, ataxia, motor deficits, and microcephaly. AS is caused by loss-of-function of the maternally inherited UBE3A gene. UBE3A is located on chromosome 15q11-13 and is biallelically expressed throughout the body but only maternally expressed in the brain due to an RNA antisense transcript that silences the paternal copy. There is currently no cure for AS, but advancements in small molecule drugs and gene therapies offer a promising approach for the treatment of the disorder. Here, we review AS and how loss-of-function of the maternal UBE3A contributes to the disorder. We also discuss the strengths and limitations of current animal models of AS. Furthermore, we examine potential small molecule drug and gene therapies for the treatment of AS and associated challenges faced by the therapeutic design. Finally, gene therapy offers the opportunity for precision medicine in AS and advancements in the treatment of this disorder can serve as a foundation for other single-gene neurodevelopmental disorders.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window.