Prevalence and long-term monitoring of humoral immunity against adeno-associated virus in Duchenne Muscular Dystrophy patients.

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans.

Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors.

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8(+) cells. IFN-γ-producing cells were also observed among effector memory CD4(+) cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.

Assessing patiromer utilization and associated serum potassium changes in US veterans with prior sodium polystyrene sulfonate exposure.

Untreated chronic hyperkalemia is associated with an increased risk of mortality. Novel potassium binders (e.g., patiromer) are new additions to the clinician's armamentarium. Prior to their approval, clinicians often considered trialing sodium polystyrene sulfonate. The study objective was to assess patiromer utilization and associated changes in serum potassium (K+) in US veterans with prior sodium polystyrene sulfonate exposure. This was a real-world observational study of US veterans with chronic kidney disease and a baseline K+ ≥ 5.1 mEq/L, initiated on patiromer between January 1, 2016, and February 28, 2021. The primary endpoints were patiromer utilization (dispensations and treatment courses), and K+ change at 30-, 91-, and 182-day follow-up (FU) intervals. Patiromer utilization was described using Kaplan-Meier probabilities and the proportion of days covered. Descriptive changes in population average K+ were obtained from a pre-post design using single-arm within-patient pre-post lab pairs and paired t tests. Two hundred five veterans met the study criteria. We observed an average of 1.25 (95% CI, 1.19-1.31) treatment courses and a median treatment duration of 64 days. Fifty veterans (24.4%) had >1 course, and 17.6% of patients remained on their initial patiromer treatment course until the end of the 180-day FU. The mean K+ value was 5.73 mEq/L (5.66-5.79) at baseline, 4.95 mEq/L (95% CI, 4.86-5.05) at the 30-day interval, 4.93 mEq/L (95% CI, 4.84-5.03) at the 91-day interval, and 4.9 mEq/L (95% CI, 4.8-4.99) at the 182-day interval. Novel potassium binders (e.g., patiromer) are newer chronic hyperkalemia management tools for clinicians. The average population K+ decreased to <5.1 mEq/L at all follow-up intervals. Patiromer appeared to be well tolerated with nearly 18% of patients remaining on their initial treatment course during the entire 180-day FU period. The median treatment duration was 64 days and approximately 24% of patients initiated a second course during FU.

A 10 patient case report on the impact of plasmapheresis upon neutralizing factors against adeno-associated virus (AAV) types 1, 2, 6, and 8.

Adeno-associated viruses (AAV) are small, nonenveloped single-stranded DNA viruses which require helper viruses to facilitate efficient replication. These recombinant viruses are some of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Nevertheless, the presence of humoral responses to the wild-type AAV common among humans is one of the limitations of in vivo transduction efficacy in humans using cognate recombinant vector. In this study, based on the serum samples that we were able to collect from various clinical situations, we studied the impact of one to five plasmapheresis (PP), at 1-5 day intervals on neutralizing factor (NAF) titers specific for AAV types 1, 2, 6, and 8 in seropositive patients with diverse pathologies and immunosuppressor treatments. We show that frequent sessions of PP result in drastic reduction of NAF specific for AAV1, 2, 6, and 8 to undetectable levels or titers <1:5, mainly when initial titers, i.e., before the first PP were ≤1:20. Altogether, these results show that the use of PP and its possible association with pharmacological immunosuppressive treatments may help to design optimal management of seropositive patients for AAV gene therapy treatments.

Real-world evaluation of patiromer utilization and its effects on serum potassium in veterans with end stage kidney disease.

Hyperkalemia (serum potassium [K+] ≥5.1) is life-threatening in patients diagnosed with end stage kidney disease (ESKD). Patiromer is approved for the treatment of hyperkalemia, although its role in hyperkalemic patients with ESKD is not well understood. This study describes real-world patiromer utilization in an ESKD population and its corresponding association with serum K+ level changes. The study population was comprised of US veterans with an outpatient dispensing of patiromer and 2 or more International Classification of Diseases diagnostic codes for ESKD. A treatment course of patiromer was defined by serial dispensing events without a 30-day gap. Patiromer utilization was described by duration, average dose, persistence, and proportion of days covered during patiromer course. Mean serum K+ values were described for baseline and 3 follow-up intervals during the 180-day follow-up period. There were 458 patients with ESKD included in the study. On average, patients had 1.24 (95% CI: 1.20-1.29) patiromer courses. Half of the population discontinued their first patiromer course within 30 days, while approximately 10% of patients remained persistent at the end of the 180-day period and 102 (22.3%) patients started a second course during the 180-day follow up period. Average serum K+ concentrations during baseline and the 3 evaluation intervals during the 180-day follow-up were 5.91 mEq/L (5.85-5.97), 4.94 mEq/L (4.86-5.03), 4.89 mEq/L (4.8-4.98) and 4.88 mEq/L (4.8-4.96). Few patients remained persistent on their initial course of patiromer at the end of follow-up, but approximately 20% of patients initiated a second treatment episode after a 30-day gap in treatment during the 180-day follow-up period. Nonetheless, average serum K+ in ESKD patients were sustainably reduced by approximately 1 mEq/L during follow-up.

Capsid-specific removal of circulating antibodies to adeno-associated virus vectors.

Neutralizing antibodies directed against adeno-associated virus (AAV) are commonly found in humans. In seropositive subjects, vector administration is not feasible as antibodies neutralize AAV vectors even at low titers. Consequently, a relatively large proportion of humans is excluded from enrollment in clinical trials and, similarly, vector redosing is not feasible because of development of high-titer antibodies following AAV vector administration. Plasmapheresis has been proposed as strategy to remove anti-AAV antibodies from the bloodstream. Although safe and relatively effective, the technology has some limitations mainly related to the nonspecific removal of all circulating IgG. Here we developed an AAV-specific plasmapheresis column which was shown to efficiently and selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma. We showed the nearly complete removal of anti-AAV antibodies from high titer purified human IgG pools and plasma samples, decreasing titers to levels that allow AAV vector administration in mice. These results provide proof-of-concept of a method for the AAV-specific depletion of neutralizing antibodies in the setting of in vivo gene transfer.

Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation.

BACKGROUND: Gene modified dendritic cells (DC) are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC) are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. METHODS: We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV) pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG), the gibbon ape leukaemia virus envelope (GaLV) or the feline endogenous virus envelope (RD114). At the same time, we evaluated transgene expression (E-GFP reporter gene) under the control of different promoters. RESULTS: We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV) under the control of phoshoglycerate kinase (PGK) and elongation factor-1 (EF1alpha) promoters (28% to 90% of E-GFP+ cells, respectively) in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5-12) described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. CONCLUSION: Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

Polymers for improving the in vivo transduction efficiency of AAV2 vectors.

BACKGROUND: Adeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency. METHODOLOGY: Here, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2. CONCLUSIONS: Our results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins.

Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors.

Adeno-associated viruses (AAVs) are small, nonenveloped single-stranded DNA viruses that require helper viruses to facilitate efficient replication. Despite the presence of humoral responses to the wild-type AAV in humans, AAV remains one of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Characterization of the IgG subclass responses to AAV and study of the prevalence of both IgG and neutralizing factors to AAV types 1, 2, 5, 6, 8, and 9 in the human population are of importance for the development of new strategies to overcome these immune responses. Natural exposure to AAV types 1, 2, 5, 6, 8, and 9 can result in the production of antibodies from all four IgG subclasses, with a predominant IgG1 response and low IgG2, IgG3, and IgG4 responses. Prevalences of anti-AAV1 and -AAV2 total IgG determined by enzyme-linked immunosorbent assay were higher (67 and 72%) than those of anti-AAV5 (40%), anti-AAV6 (46%), anti-AAV8 (38%), and anti-AAV9 (47%). Furthermore, data showed that cross-reactions are important. The two highest neutralizing factor seroprevalences were observed for AAV2 (59%) and AAV1 (50.5%) and the lowest were observed for AAV8 (19%) and AAV5 (3.2%). Vectors based on AAV5, AAV8, and AAV9 may have an advantage for gene therapy in humans. Furthermore, among individuals seropositive for AAV5, AAV8, and AAV9, about 70-100% present low titers. Better characterization of the preexisting humoral responses to the AAV capsid and cross-reactivity will allow development of new strategies to circumvent AAV acquired immune responses.