Realistic paths to gene therapy for the X-linked bleeding disorder hemophilia started to materialize in the mid 1990s, resulting in disease correction in small and large animal models. Out of a diversity of approaches, in vivo adeno-associated viral (AAV) gene transfer to hepatocytes emerged as the most promising strategy, eventually forming the basis for multiple advanced clinical trials and regulatory approval of two products for the treatment of hemophilia B (coagulation factor IX deficiency) and one for hemophilia A (factor VIII deficiency). Ideally, gene therapy is effective with a single administration, thus providing therapeutic factor levels over a period of years, without the need for frequent injections. Overcoming multiple obstacles, some not predicted by preclinical studies, sustained partial to complete correction of coagulation for several years to an entire decade has now been documented in patients, with observation ongoing. A hyperactive form of FIX improved efficacy in hemophilia B, and superior engineered variants of FVIII are emerging. Nonetheless, challenges remain, including pre-existing immunity to AAV capsids, toxicities, inter-patient variability in response to treatment, and difficulty in obtaining durable therapeutic expression of FVIII. In alternative approaches, in vivo gene editing and ex vivo gene therapies targeting hemopoietic cells are in development.

Voretigene neparvovec-rzyl (Luxturna) is an AAV2 vector (AAV2-hRPE65v2) that expresses a cDNA encoding the human retinal pigment epithelium-specific 65 kDa protein (RPE65). It has been approved for the treatment of visual deficits associated with biallelic mutations in human RPE65 in the US, European Union (EU), and multiple other countries. To achieve regulatory approval, it was necessary to validate an assay demonstrating its biological activity or potency. The assay measures AAV2hRPE65v2 transduction in HEK293 cells and the subsequent biological activity of the vector-encoded RPE65 protein in cell lysates. RPE65 converts all-trans-retinol to 11-cis-retinol, which is quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The assay was validated for seven characteristics, namely system and sample suitability, specificity, linearity, precision, relative accuracy, range, and robustness. The validated assay can be used to confirm the relative potency levels of different lots of Luxturna in the range of 50%-150% of a reference standard (defined as 100% potent). This represents the first report of validation studies supporting an in vitro cell-based relative potency assay for an AAV vector, which was used to evaluate lot-to-lot consistency, stability, and comparability following manufacturing changes and to successfully launch Luxturna, the first gene therapy approved in the US for a genetic disease.

BACKGROUND:Treatment with fidanacogene elaparvovec, a recombinant adeno-associated virus (AAV) vector developed for the treatment of hemophilia B, led to sustained expression of the high-activity factor IX variant (FIX-R338L, or FIX-Padua) in a phase 1-2a study. The long-term safety and efficacy of this treatment are not known. METHODS:In a 12-month study, 15 participants with severe or moderately severe hemophilia B (factor IX coagulant activity, ≤2% of the normal value) received fidanacogene elaparvovec at a dose of 5×1011 vector genomes (vg) per kilogram of body weight; thereafter, participants could enroll in a 5-year follow-up study. Safety end points included adverse events and changes in laboratory measures. Efficacy end points included the annualized rate of treated bleeding events (annualized bleeding rate) and factor IX activity. RESULTS:A total of 14 participants provided consent and completed at least 3 years of follow-up (median, 5.5; range 3 to 6); participation was ongoing among 8 at the data cutoff. None of the participants reported treatment-related adverse events after year 1. Throughout follow-up, nine serious adverse events were noted in 4 participants; none were thrombotic or treatment-related. No factor IX inhibitors were detected. Throughout follow-up, mean factor IX activity was in the mild hemophilia range; the mean annualized bleeding rate was less than 1, and 10 participants had no treated bleeding episodes. Surveillance liver ultrasounds obtained from year 1 onward showed no evidence of cancer but showed steatosis in 4 participants who had weight gain and elevated aminotransferase levels (maximum alanine aminotransferase level, 77 U per liter). One participant with a history of hepatitis C, hepatitis B, human immunodeficiency virus infection, and an elevated body-mass index had progression of underlying advanced liver fibrosis. A total of 13 surgical procedures were performed in 8 participants; exogenous factor IX was administered for 10 procedures, and no associated unexpected bleeding complications occurred. CONCLUSIONS:Fidanacogene elaparvovec was associated with no or only low-grade adverse effects over a period of 3 to 6 years. Efficacy was maintained in the long term at 5×1011 vg per kilogram, one of the lowest intravenous doses of AAV used for any indication. (Funded by Pfizer; ClinicalTrials.gov number, NCT03307980.).

ABSTRACT:The US Food and Drug Administration (FDA)'s authorization of etranacogene dezaparvovec (Hemgenix) is a significant milestone, constituting not only the first FDA approval of a gene therapy for hemophilia but also the first approval of a liver-targeted adeno-associated virus vector gene therapy. This review summarizes the nonclinical studies and clinical development that supported regulatory clearance. Similar to other gene therapies for single gene disorders, both the short-term safety and the phenotypic improvement were unequivocal, justifying the modest-sized safety and efficacy database, which included 57 participants across the phase 2b (3 participants) and phase 3 (54 participants) studies. The most common adverse reactions included liver enzyme elevation, headache, flu-like symptoms, infusion-related reactions, creatine kinase elevation, malaise, and fatigue; these were mostly transient. One participant had hepatocellular carcinoma on a study-mandated liver ultrasound conducted 1 year after vector infusion; molecular analysis of the resected tumor showed no evidence of vector-related insertional mutagenesis as the etiology. A remarkable 96% of participants in the phase 3 trial were able to stop factor IX (FIX) prophylaxis, with the study demonstrating noninferiority to FIX prophylaxis in terms of the primary end point, annualized bleeding rate. Key secondary end points such as the annualized infusion rate, which declined by 97%, and the plasma FIX activity level at 18 months after infusion, with least squares mean increase of 34.3 percentage points compared with baseline, were both clinically and statistically significant. The FDA's landmark approval of Hemgenix as a pioneering treatment for hemophilia stands on the shoulders of >20 years of gene therapy clinical research and heralds a promising future for genomic medicines.

Gene therapy, including both in vivo gene transfer and therapy with gene-modified cells, has moved in recent years from the realm of investigation to marketed products and is now one of the fastest growing classes of therapeutics. Two articles recently published in Molecular Therapy seek to provide a view of ongoing and recent activities in the space. 1 Chancellor D. Barrett D. Nguyen-Jatkoe L. Millington S. Eckhardt F. The state of cell and gene therapy in 2023. Mol. Ther. 2023; 31: 3376-3388 Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar ,2 Palomo G.M. Pose-Boirazian T. Naumann-Winter F. Costa E. Duarte D.M. Kalland M.E. Malikova E. Matusevicius D. Vitezic D. Larsson K. et al. The European landscape for gene therapies in orphan diseases: 6-year experience with the EMA Committee for Orphan Medicinal Products. Mol. Ther. 2023; 31: 3414-3423 Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Though they use different methodologies and indeed begin from altogether different definitions of gene therapy, they reach some of the same conclusions or at least have similar implications. Both agree, for example, that there is a great deal of activity in the space, that the pace of approvals has accelerated in recent years, and that the pipeline is robust.

Given the therapeutic potential of supplying a normal copy of a mutant gene to the correct target tissue, gene therapy holds extraordinary promise for the treatment of genetic disease. Like other novel classes of therapeutics however, gene therapies must overcome a range of clinical, regulatory, and manufacturing hurdles to reach regulatory approval. This paper reviews key aspects of clinical trial design, development, and evaluation of a novel primary end point, and regulatory interactions that resulted in the first approval by the U.S. Food and Drug Administration (FDA) of an adeno-associated virus (AAV) gene therapy product.

Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our secondgeneration expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 x 1012 vg/kg FVIII antigen levels of 13% +/- 2% and 22% +/- 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic.

Limited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX‐Padua gene therapy.

Purpose: To determine whether functional vision and visual function improvements after voretigene neparvovec (VN; Luxturna [Spark Therapeutics, Inc]) administration in patients with biallelic RPE65 mutation-associated inherited retinal disease are maintained at 3 to 4 years and to review safety outcomes. Design: Open-label, randomized, controlled phase 3 trial. Participants: Thirty-one individuals were enrolled and randomized 2:1 to intervention (n = 21) or control (n = 10). One participant from each group withdrew before, or at, randomization. Methods: Patients in the original intervention (OI) group received bilateral subretinal VN injections. Delayed intervention (DI) patients served as control participants for 1 year then received VN. Main Outcome Measures: Change from injection baseline in bilateral performance on the multiluminance mobility test (MLMT), a measure of ambulatory navigation, and change from injection baseline in full-field light sensitivity threshold white light, visual field (VF), and visual acuity (VA). Results: Mean bilateral MLMT change scores at year 4 for OI patients and year 3 for DI patients were 1.7 and 2.4, respectively, with 71% of patients with a year 3 visit able to pass MLMT at the lowest light level. Mean change in full-field light sensitivity threshold white light, averaged over both eyes at year 4 for OI patients and year 3 for DI patients, was -1.90 log(10)(cd.s/m(2)) and -2.91 log(10)(cd.s/m(2)), respectively. Mean change in Goldmann kinetic VF III4e sum total degrees, averaged across both eyes, was 197.7 at year 4 for OI patients and 157.9 at year 3 for DI patients. Mean change in VA (Holladay scale), averaged across both eyes, was -0.003 logarithm of the minimum angle of resolution (logMAR) at year 4 for OI patients and -0.06 logMAR at year 3 for DI patients. One OI patient experienced retinal detachment at approximately year 4 that impacted VA for the OI group. No product-related serious adverse events (AEs) occurred, nor did any deleterious immune responses. Conclusions: Improvements in ambulatory navigation, light sensitivity, and VF were consistent in both intervention groups. Overall, improvements were maintained up to 3 to 4 years, with ongoing observation. The safety profile of VN was consistent with vitrectomy and the subretinal injection procedure and was similar between intervention groups, with no product-related serious AEs reported. (C) 2021 by the American Academy of Ophthalmology.

To determine whether functional vision and visual function improvements after voretigene neparvovec (VN; Luxturna [Spark Therapeutics, Inc]) administration in patients with biallelic RPE65 mutation-associated inherited retinal disease are maintained at 3 to 4 years and to review safety outcomes.Open-label, randomized, controlled phase 3 trial.Thirty-one individuals were enrolled and randomized 2:1 to intervention (n = 21) or control (n = 10). One participant from each group withdrew before, or at, randomization.Patients in the original intervention (OI) group received bilateral subretinal VN injections. Delayed intervention (DI) patients served as control participants for 1 year then received VN.Change from injection baseline in bilateral performance on the multiluminance mobility test (MLMT), a measure of ambulatory navigation, and change from injection baseline in full-field light sensitivity threshold white light, visual field (VF), and visual acuity (VA).Mean bilateral MLMT change scores at year 4 for OI patients and year 3 for DI patients were 1.7 and 2.4, respectively, with 71% of patients with a year 3 visit able to pass MLMT at the lowest light level. Mean change in full-field light sensitivity threshold white light, averaged over both eyes at year 4 for OI patients and year 3 for DI patients, was -1.90 log10(cd.s/m2) and -2.91 log10(cd.s/m2), respectively. Mean change in Goldmann kinetic VF III4e sum total degrees, averaged across both eyes, was 197.7 at year 4 for OI patients and 157.9 at year 3 for DI patients. Mean change in VA (Holladay scale), averaged across both eyes, was -0.003 logarithm of the minimum angle of resolution (logMAR) at year 4 for OI patients and -0.06 logMAR at year 3 for DI patients. One OI patient experienced retinal detachment at approximately year 4 that impacted VA for the OI group. No product-related serious adverse events (AEs) occurred, nor did any deleterious immune responses.Improvements in ambulatory navigation, light sensitivity, and VF were consistent in both intervention groups. Overall, improvements were maintained up to 3 to 4 years, with ongoing observation. The safety profile of VN was consistent with vitrectomy and the subretinal injection procedure and was similar between intervention groups, with no product-related serious AEs reported.

Goal To determine whether ambulatory navigation, light sensitivity, and visual field (VF) improvements 1 year after voretigene neparvovec (VN) administration in patients with biallelic RPE65 mutation-associated inherited retinal dystrophy (IRD) are maintained at 5 years and review safety outcomes over the entire period. Methods This is an open label, randomized, controlled Phase III trial performed at 2 sites in the United States. Patients were randomized to either original intervention (OI: bilateral subretinal VN at baseline; n=20) or delayed intervention (DI: VN after 1 year; n=9). The primary endpoint was bilateral performance on the Multi-Luminance Mobility Test (MLMT) at 7 standard light levels as measured by a change in score. Additional endpoints were full-field light sensitivity threshold (FST) testing, visual acuity (VA), and Goldmann kinetic VF (GVF), each averaged over both eyes. Safety outcomes included adverse event reporting, laboratory testing, and changes in physical and ophthalmic examinations. Results For OI patients at Year 5 (n=18) and DI patients at Year 4 (n=8), the mean (standard deviation) MLMT bilateral light level score change was 1.6 (1.1) and 2.4 (1.5) levels, respectively, compared with baseline. Subsequent to the 1-year outcomes, a change of 1 light level occurred in 6 patients (none were below pre-treatment performance) and no change in the remaining 20 (N=26). Mean change in white light FST in log10 (cd.s/m2) averaged over both eyes was −2.02 (1.45) log10 at Year 5 for OI patients (n=17) and −2.58 (1.04) log10 at Year 4 for DI patients (n=8). Mean change in VA (Holladay Scale) averaged over both eyes (logMAR was −0.00 (0.64) at Year 5 for OI patients (n=18) and −0.06 (0.26) at Year 4 for DI patients (n=8). Mean change in GVF III4e sum total degrees averaged over both eyes was 166.6 (208.7) at Year 5 for OI patients (n=15) and 178.8 (241.9) at Year 4 for DI patients (n=8). Five years after treatment, the safety profile (N=29) was consistent with vitrectomy and subretinal injection procedure with 2 reports of cataract, 1 of ptosis, and 1 new report of retinal detachment since the last update. No deleterious immune responses were reported. Conclusion Improvements in ambulatory navigation, light sensitivity, and VF are maintained for at least 5 years after VN administration in most OI patients. Improvements in DI patients were consistent with those observed in OI patients. The safety profile of VN is consistent with the administration procedure.

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n = 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8 x 10(10) to 2 x 10(12) vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n = 4) was followed for 12-15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum alpha-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years postAAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy.

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.

Much of what is understood about specific coagulation proteins has emerged from the careful study of hereditary disorders of blood coagulation. Haemophilia is a familial X-linked disorder due to deficiency of either factor VIII (haemophilia A) or factor IX (haemophilia B), components of the intrinsic enzymatic complex that activates factor X. The severity of the disease correlates with predicted concentrations of activated factor protein, and those with activity levels below 1% are defined as having severe disease....