Casein kinase 2 activity is a host restriction factor for AAV transduction.

So far, the mechanisms that impede AAV transduction, especially in the human heart, are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs), iPSC-derived cardiac fibroblasts (iPSC-CFs), and primary endothelial cells to model vector-host interactions. Through phosphoproteome analysis we established that casein kinase 2 (CK2) signaling is one of the most significantly affected pathways upon AAV exposure. Transient inhibition of CK2 activity substantially enhanced the transduction rate of AAV2, AAV6, and AAV9 in all tested cell types. In particular, CK2 inhibition improved the trafficking of AAVs through the cytoplasm, impaired DNA damage response through destabilization of MRE11, and altered the RNA processing pathways, which were also highly responsive to AAV transduction. Also, it augmented transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. In summary, the present study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, which may translate to improving the outcome of AAV-based therapies in the future.

Endothelial-to-mesenchymal transition enhances permissiveness to AAV vectors in cardiac endothelial cells.

A major obstacle in inducing therapeutic angiogenesis in the heart is inefficient gene transfer to endothelial cells (ECs). Here, we identify compounds able to enhance the permissiveness of cardiac ECs to adeno-associated virus (AAV) vectors, which stand as ideal tools for in vivo gene delivery. We screened a library of >1,500 US Food and Drug Administration (FDA)-approved drugs, in combination with AAV vectors, in cardiac ECs. Among the top drugs increasing AAV-mediated transduction, we found vatalanib, an inhibitor of multiple tyrosine kinase receptors. The increased AAV transduction efficiency by vatalanib was paralleled by induction of the endothelial-to-mesenchymal transition, as documented by decreased endothelial and increased mesenchymal marker expression. Induction of the endothelial-to-mesenchymal transition by other strategies similarly increased EC permissiveness to AAV vectors. In vivo injection of AAV vectors in the heart after myocardial infarction resulted in the selective transduction of cells undergoing the endothelial-to-mesenchymal transition, which is known to happen transiently after cardiac ischemia. Collectively, these results point to the endothelial-to-mesenchymal transition as a mechanism for improving AAV transduction in cardiac ECs, with implications for both basic research and the induction of therapeutic angiogenesis in the heart.

In vivo base editing of a pathogenic Eif2b5 variant improves vanishing white matter phenotypes in mice.

Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models. Using adeno-associated viral vectors, we delivered intein-split adenine base editors into the cerebral ventricles of newborn VWM mice, resulting in 45.9% ± 5.9% correction of the Eif2b5R191H variant in the cortex. Treatment slightly increased mature astrocyte populations and partially recovered the integrated stress response (ISR) in female VWM animals. This led to notable improvements in bodyweight and grip strength in females; however, locomotor disabilities were not rescued. Further molecular analyses suggest that more precise editing (i.e., lower rates of bystander editing) as well as more efficient delivery of the base editors to deep brain regions and oligodendrocytes would have been required for a broader phenotypic rescue. Our study emphasizes the potential, but also identifies limitations, of current in vivo base-editing approaches for the treatment of VWM or other leukodystrophies.

Targeted gene therapy for rare genetic kidney diseases.

Chronic kidney disease (CKD) is one of the leading causes of mortality worldwide because of kidney failure and the associated challenges of its treatment including dialysis and kidney transplantation. About one-third of CKD cases are linked to inherited monogenic factors, making them suitable for potential gene therapy interventions. However, the intricate anatomical structure of the kidney poses a challenge, limiting the effectiveness of targeted gene delivery to the renal system. In this review, we explore the progress made in the field of targeted gene therapy approaches and their implications for rare genetic kidney disorders, examining preclinical studies and prospects for clinical application. In vivo gene therapy is most commonly used for kidney-targeted gene delivery and involves administering viral and non-viral vectors through various routes such as systemic, renal vein and renal arterial injections. Small nucleic acids have also been used in preclinical and clinical studies for treating certain kidney disorders. Unexpectedly, hematopoietic stem and progenitor cells have been used as an ex vivo gene therapy vehicle for kidney gene delivery, highlighting their ability to differentiate into macrophages within the kidney, forming tunneling nanotubes that can deliver genetic material and organelles to adjacent kidney cells, even across the basement membrane to target the proximal tubular cells. As gene therapy technologies continue to advance and our understanding of kidney biology deepens, there is hope for patients with genetic kidney disorders to eventually avoid kidney transplantation.

Enhancement of adeno-associated virus serotype 6 transduction into T cells with cell-penetrating peptides.

BACKGROUND: Adeno-associated viruses (AAVs) are gaining interest in the development of cellular immunotherapy. Compared to other viral vectors, AAVs can reduce the risk of insertional oncogenesis. AAV serotype 6 (AAV6) shows the highest efficiency for transducing T cells. Nevertheless, a multiplicity of infection (MOI) of up to one million viral genomes per cell is required to transduce the target cells effectively. Cell-penetrating peptides (CPPs) are short, positively charged peptides that easily translocate the plasma membranes and can facilitate the cellular uptake of a wide variety of cargoes, including small molecules, nucleic acids, drugs, proteins and viral vectors. METHODS: The present study evaluated five CPPs (Antp, TAT-HA2, LAH4, TAT1 and TAT2) on their effects on enhancing transduction of AAV6 packaging a green fluorescent protein transgene into Jurkat T cell line. RESULTS: Vector incubation with peptides TAT-HA2 and LAH4 at a final concentration of 0.2 mm resulted in an approximately two-fold increase in transduced cells. At the lowest MOI tested (1.25 × 104 ), using LAH4 resulted in a 10-fold increase in transduction efficiency. The peptide LAH4 increased the uptake of AAV6 viral particles in both Jurkat cells and mouse primary T cells. Regardless of the large size of the AAV6-LAH4 complexes, their internalization does not appear to depend on macropinocytosis. CONCLUSIONS: Overall, the present study reports an approach to significantly improve the delivery of transgenes into T cells using AAV6 vectors. Notably, the peptides TAT-HA2 and LAH4 contribute to improving the use of AAV6 as a gene delivery vector for the engineering of T cells.

Preclinical Milestones in MECP2 Gene Transfer for Treating Rett Syndrome.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). After gene transfer in mice, exogenous MeCP2 expression must be regulated to avoid dose-dependent toxicity. SUMMARY: The preclinical gene therapy literature for treating RTT illustrates a duly diligent progression that begins with proof-of-concept studies and advances toward the development of safer, regulated MECP2 viral genome designs. This design progression was partly achieved through international collaborative studies. In 2023, clinicians administered investigational gene therapies for RTT to patients a decade after the first preclinical gene therapy publications for RTT (clinical trial numbers NCT05606614 and NCT05898620). As clinicians take on a more prominent role in MECP2 gene therapy research, preclinical researchers may continue to test more nuanced hypotheses regarding the safety, efficacy, and mechanism of MECP2 gene transfer. KEY MESSAGE: This review summarizes the history of preclinical MECP2 gene transfer for treating RTT and acknowledges major contributions among colleagues in the field. The first clinical injections are a shared milestone.

Integration and the risk of liver cancer-Is there a real risk?

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.

Gene therapy for ultrarare diseases: a geneticist's perspective.

Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies.

BASIS: BioAnalysis SEDFIT integrated software for cGMP analysis of SV-AUC data.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has long been an important method for characterization of antibody therapeutics. Recently, SV-AUC has experienced a wave of new interest and usage from the gene and cell therapy industry, where SV-AUC has proven itself to be the "gold standard" analytical approach for determining capsid loading ratios for adeno-associated virus (AAV) and other viral vectors. While other more common approaches have existed in the realm of cGMP-compliant techniques for years, SV-AUC has long been used strictly for characterization, but not for release testing. This manuscript describes the challenges faced in bringing SV-AUC to a cGMP environment and describes a new program, "BASIS", which allows for 21 CFR Part 11-compliant data handling and data analysis using the well-known and frequently cited SEDFIT analysis software.

Preclinical studies in Krabbe disease: A model for the investigation of novel combination therapies for lysosomal storage diseases.

Krabbe disease (KD) is a lysosomal storage disease (LSD) caused by mutations in the galc gene. There are over 50 monogenetic LSDs, which largely impede the normal development of children and often lead to premature death. At present, there are no cures for LSDs and the available treatments are generally insufficient, short acting, and not without co-morbidities or long-term side effects. The last 30 years have seen significant advances in our understanding of LSD pathology as well as treatment options. Two gene therapy-based clinical trials, NCT04693598 and NCT04771416, for KD were recently started based on those advances. This review will discuss how our knowledge of KD got to where it is today, focusing on preclinical investigations, and how what was discovered may prove beneficial for the treatment of other LSDs.

Organoid transduction using recombinant adeno-associated viral vectors: Challenges and opportunities.

Cellular 3D structures, for example, organoids, are an excellent model for studying and developing treatments for various diseases, including hereditary ones. Therefore, they are increasingly being used in biomedical research. From the point of view of safety and efficacy, recombinant adeno-associated viral (rAAV) vectors are currently most in demand for the delivery of various transgenes for gene replacement therapy or other applications. The delivery of transgenes using rAAV vectors to various types of organoids is an urgent task, however, it is associated with a number of problems that are discussed in this review. Cellular heterogeneity and specifics of cultivation of 3D structures determine the complexity of rAAV delivery and are sometimes associated with low transduction efficiency. This review surveys the main ways to solve emerging problems and increase the efficiency of transgene delivery using rAAVs to organoids. A clear understanding of the stage of development of the organoid, its cellular composition and the presence of surface receptors will allow obtaining high levels of organoid transduction with existing rAAV vectors.

The Road towards Gene Therapy for X-Linked Juvenile Retinoschisis: A Systematic Review of Preclinical Gene Therapy in Cell-Based and Rodent Models of XLRS.

X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE's risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives.

Targeted Delivery of Chimeric Antigen Receptor into T Cells via CRISPR-Mediated Homology-Directed Repair with a Dual-AAV6 Transduction System.

CAR-T cell therapy involves genetically engineering T cells to recognize and attack tumour cells by adding a chimeric antigen receptor (CAR) to their surface. In this study, we have used dual transduction with AAV serotype 6 (AAV6) to integrate an anti-CD19 CAR into human T cells at a known genomic location. The first viral vector expresses the Cas9 endonuclease and a guide RNA (gRNA) targeting the T cell receptor alpha constant locus, while the second vector carries the DNA template for homology-mediated CAR insertion. We evaluated three gRNA candidates and determined their efficiency in generating indels. The AAV6 successfully delivered the CRISPR/Cas9 machinery in vitro, and molecular analysis of the dual transduction showed the integration of the CAR transgene into the desired location. In contrast to the random integration methods typically used to generate CAR-T cells, targeted integration into a known genomic locus can potentially lower the risk of insertional mutagenesis and provide more stable levels of CAR expression. Critically, this method also results in the knockout of the endogenous T cell receptor, allowing target cells to be derived from allogeneic donors. This raises the exciting possibility of "off-the-shelf" universal immunotherapies that would greatly simplify the production and administration of CAR-T cells.

AAV analysis by sedimentation velocity analytical ultracentrifugation: beyond empty and full capsids.

The recent surge of therapeutic interest in recombinant adeno-associated viral (AAV) vectors for targeted DNA delivery has brought analytical ultracentrifugation (AUC) into the spotlight. A major concern during formulation of AAV therapeutics is purity of the active species (DNA-containing capsid, or "filled capsids"). Insertion of DNA into AAV is not a highly efficient process; thus, a significant amount of empty and partial/intermediate AAV molecules may exist. Recent guidance from the FDA includes limiting the presence of empty AAV capsids and other impurities to reduce immunotoxicity. While chromatographic techniques (SEC, SEC-MALS, AEX) are often used for empty and full capsid quantitation due to the ease of accessibility and familiarity among most biochemists, the resolution and sensitivity attained by sedimentation velocity (SV-AUC) in the formulation buffer and purification buffers is unmatched. Approaches for using SV-AUC to determine the empty-to-full capsid ratio have already been discussed by others; however, in this report, we focus on the importance of characterizing other impurities, such as free DNA, partially filled capsids, and aggregates that are recognized as species of concern for immunotoxicity. We also demonstrate the usefulness of applying multiple analyses (e.g., c(s), g(s*), WDA) in confirming the presence of and determining the hydrodynamic parameters of these various species.

Sphingosine-1-phosphate transporter spinster homolog 2 is essential for iron-regulated metastasis of hepatocellular carcinoma.

Iron dyshomeostasis is associated with hepatocellular carcinoma (HCC) development. However, the role of iron in HCC metastasis is unknown. This study aimed to elucidate the underlying mechanisms of iron's enhancement activity on HCC metastasis. In addition to the HCC cell lines and clinical samples in vitro, iron-deficient (ID) mouse models were generated using iron-free diet and transferrin receptor protein knockout, followed by administration of HCC tumors through either orthotopic or ectopic route. Clinical metastatic HCC samples showed significant ID status, accompanied by overexpression of sphingosine-1-phosphate transporter spinster homolog 2 (SPNS2). Mechanistically, ID increased SPNS2 expression, leading to HCC metastasis in both cell cultures and mouse models. ID not only altered the anti-tumor immunity, which was indicated by phenotypes of lymphatic subsets in the liver and lung of tumor-bearing mice, but also promoted HCC metastasis in a cancer cell autonomous manner through the SPNS2. Since germline knockout of globe SPNS2 showed significantly reduced HCC metastasis, we further developed hepatic-targeting recombinant adeno-associated virus vectors to knockdown SPNS2 expression and to inhibit iron-regulated HCC metastasis. Our observation indicates the role of iron in HCC pulmonary metastasis and suggests SPNS2 as a potential therapeutic target for the prevention of HCC pulmonary metastasis.

ULK overexpression mitigates motor deficits and neuropathology in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal neurodegenerative disorder clinically characterized by prominent ataxia. It is caused by an expansion of a CAG trinucleotide in ATXN3, translating into an expanded polyglutamine (polyQ) tract in the ATXN3 protein, that becomes prone to misfolding and aggregation. The pathogenesis of the disease has been associated with the dysfunction of several cellular mechanisms, including autophagy and transcription regulation. In this study, we investigated the transcriptional modifications of the autophagy pathway in models of MJD and assessed whether modulating the levels of the affected autophagy-associated transcripts (AATs) would alleviate MJD-associated pathology. Our results show that autophagy is impaired at the transcriptional level in MJD, affecting multiple AATs, including Unc-51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2), two homologs involved in autophagy induction. Reinstating ULK1/2 levels by adeno-associated virus (AAV)-mediated gene transfer significantly improved motor performance while preventing neuropathology in two in vivo models of MJD. Moreover, in vitro studies showed that the observed positive effects may be mainly attributed to ULK1 activity. This study provides strong evidence of the beneficial effect of overexpression of ULK homologs, suggesting these as promising instruments for the treatment of MJD and other neurodegenerative disorders.

Dynamic Changes to the Skeletal Muscle Proteome and Ubiquitinome Induced by the E3 Ligase, ASB2ß.

Ubiquitination is a posttranslational protein modification that has been shown to have a range of effects, including regulation of protein function, interaction, localization, and degradation. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2ß, is downregulated in models of muscle growth and that overexpression ASB2ß is sufficient to induce muscle atrophy. To gain insight into the effects of increased ASB2ß expression on skeletal muscle mass and function, we used liquid chromatography coupled to tandem mass spectrometry to investigate ASB2ß-mediated changes to the skeletal muscle proteome and ubiquitinome, via a parallel analysis of remnant diGly-modified peptides. The results show that viral vector-mediated ASB2ß overexpression in murine muscles causes progressive muscle atrophy and impairment of force-producing capacity, while ASB2ß knockdown induces mild muscle hypertrophy. ASB2ß-induced muscle atrophy and dysfunction were associated with the early downregulation of mitochondrial and contractile protein abundance and the upregulation of proteins involved in proteasome-mediated protein degradation (including other E3 ligases), protein synthesis, and the cytoskeleton/sarcomere. The overexpression ASB2ß also resulted in marked changes in protein ubiquitination; however, there was no simple relationship between changes in ubiquitination status and protein abundance. To investigate proteins that interact with ASB2ß and, therefore, potential ASB2ß targets, Flag-tagged wild-type ASB2ß, and a mutant ASB2ß lacking the C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2ß interactome. ASB2ß was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, our studies have identified novel putative ASB2ß target substrates that warrant further investigation. These findings provide novel insight into the complexity of proteome and ubiquitinome changes that occur during E3 ligase-mediated skeletal muscle atrophy and dysfunction.

Efficient removal of antibodies to adeno-associated viruses by immunoadsorption.

BACKGROUND: Gene therapies based on adeno-associated viruses (AAV) are a therapeutic option to successfully treat monogenetic diseases. However, the influence of pre-existing immunity to AAV can compromise the application of AAV gene therapy, most notably by the presence of neutralizing antibodies (NAb) to AAV. METHODS: In the following study, we investigated to what extent the treatment by immunoadsorption (IA) would reduce the levels of human anti-AAV antibodies to AAV2 and AAV5. To that end, we screened blood sera from 40 patients receiving IA treatment because of underlying autoimmune disease or transplant rejection, with detectable AAV-antibodies in 23 patients (22 by NAb detection, and 1 additionally by anti-AAV5 ELISA analysis). RESULTS: Our results show that IA efficiently depleted anti-AAV2 NAb with a mean reduction of 3.92 ± 1.09 log2 titer steps (93.4%) after three to five single IA treatments, 45% of seropositive subjects had an anti-AAV2 titer below the threshold titer of 1:5 after the IA treatment series. Anti-AAV5 NAb were reduced to below the threshold titer of 1:5 in all but one of five seropositive subjects. Analysis of total anti-AAV5 antibodies by ELISA demonstrated an anti-AAV5 antibody reduction over the IA treatment series of 2.67 ± 1.16 log2 titer steps (84.3%). CONCLUSION: In summary, IA may represent a safe strategy to precondition patients with pre-existing anti-AAV antibodies to make this population eligible for an effective AAV-based gene therapy.

Prevalence of Neutralizing Antibodies against Adeno-Associated Virus Serotypes 1, 2, and 9 in Non-Injected Latin American Patients with Heart Failure-ANVIAS Study.

Neutralizing antibody (NAb) activity against the viral capsid of adeno-associated viral (AAV) vectors decreases transduction efficiency, thus limiting transgene expression. Several reports have mentioned a variation in NAb prevalence according to age, AAV serotype, and, most importantly, geographic location. There are currently no reports specifically describing the anti-AAV NAb prevalence in Latin America. Here, we describe the prevalence of NAb against different serotypes of AAV vectors (AAV1, AAV2, and AAV9) in Colombian patients with heart failure (HF) (referred to as cases) and healthy individuals (referred to as controls). The levels of NAb were evaluated in serum samples of 60 subjects from each group using an in vitro inhibitory assay. The neutralizing titer was reported as the first dilution inhibiting ≥50% of the transgene signal, and the samples with neutralizing titers at ≥1:50 dilution were considered positive. The prevalence of NAb in the case and control groups were similar (AAV2: 43% and 45%, respectively; AAV1 33.3% in each group; AAV9: 20% and 23.2%, respectively). The presence of NAb for two or more of the serotypes analyzed was observed in 25% of the studied samples, with the largest amount in the positive samples for AAV1 (55-75%) and AAV9 (93%), suggesting serial exposures, cross-reactivity, or coinfection. Moreover, patients in the HF group exhibited more common combined seropositivity for NAb against AAV1 d AAV9 than those in the control group (91.6% vs. 35.7%, respectively; p = 0.003). Finally, exposure to toxins was significantly associated with the presence of NAb in all regression models. These results constitute the first report of the prevalence of NAb against AAV in Latin America, being the first step to implementing therapeutic strategies based on AAV vectors in this population in our region.

Biochemical Correction of GM2 Ganglioside Accumulation in AB-Variant GM2 Gangliosidosis.

GM2 gangliosidosis is a group of genetic disorders that result in the accumulation of GM2 ganglioside (GM2) in brain cells, leading to progressive central nervous system (CNS) atrophy and premature death in patients. AB-variant GM2 gangliosidosis (ABGM2) arises from loss-of-function mutations in the GM2 activator protein (GM2AP), which is essential for the breakdown of GM2 in a key catabolic pathway required for CNS lipid homeostasis. In this study, we show that intrathecal delivery of self-complementary adeno-associated virus serotype-9 (scAAV9) harbouring a functional human GM2A transgene (scAAV9.hGM2A) can prevent GM2 accumulation in in GM2AP-deficient mice (Gm2a-/- mice). Additionally, scAAV9.hGM2A efficiently distributes to all tested regions of the CNS within 14 weeks post-injection and remains detectable for the lifespan of these animals (up to 104 weeks). Remarkably, GM2AP expression from the transgene scales with increasing doses of scAAV9.hGM2A (0.5, 1.0 and 2.0 × 1011 vector genomes (vg) per mouse), and this correlates with dose-dependent correction of GM2 accumulation in the brain. No severe adverse events were observed, and comorbidities in treated mice were comparable to those in disease-free cohorts. Lastly, all doses yielded corrective outcomes. These data indicate that scAAV9.hGM2A treatment is relatively non-toxic and tolerable, and biochemically corrects GM2 accumulation in the CNS-the main cause of morbidity and mortality in patients with ABGM2. Importantly, these results constitute proof-of-principle for treating ABGM2 with scAAV9.hGM2A by means of a single intrathecal administration and establish a foundation for future preclinical research.