Molecular Therapy-Methods & Clinical Development最新文献

[This corrects the article DOI: 10.1016/j.omtm.2025.101504.].

Accurate quantification and characterization of recombinant adeno-associated virus (rAAV) capsid proteins are critical for evaluating product quality and safety, ensuring batch consistency, and informing process development of their manufacture. The capsid consists of three proteins derived from the same gene, and while the mean capsid stoichiometry is nominally 1:1:10 (VP1:VP2:VP3), capsids with different stoichiometries exist. Recent studies show that variations in the capsid stoichiometry can impact vector infectivity. Here, a mass spectrometry (MS)-based method was developed to quantify VP1, VP2, and VP3 in rAAV9 capsids and determine stoichiometry. Additionally, the methodology delivers precise measurement of total capsid content and provides a greater depth of information than traditional ELISA capsid titer measurements. The method could be further refined as a reference method to standardize measurements and assign values to reference materials. Host cell proteins consistent with other findings reported in the literature were also identified and reported. The consistent detection of these host cell proteins across different studies highlights their potential relevance to gene therapy products and the importance of their monitoring. Our report exhibits the utility of MS for precise rAAV characterization and presents the first approach to using MS for the standardized measurement of rAAV across different drug products.

The multi-attribute method (MAM), a mass spectrometry technique for quantifying amino acid modifications at the peptide level, is becoming a prominent analytical tool in the development of biotherapeutics. The method has promise for adeno-associated virus (AAV) therapeutics, where capsid protein modifications have been directly linked to reduced transduction efficiency. Given this link, a robust and precise procedure to quantitate capsid modifications would be beneficial for implementation throughout biotherapeutic development. Herein, an AAV product was characterized, and capsid sequence liabilities were identified. A peptide map MAM method was developed to quantitate select sites of modifications and was validated according to ICH Q2(R2). Through this exercise, the method was demonstrated to be suitable to quantitate several sites of deamidation and the method was applied during stability, process development, and product comparability studies. Additionally, preliminary data demonstrated that the method was not limited to monitoring deamidation but also could be applied to other post-translational and chemical modifications.

Tuberous sclerosis complex (TSC) is a dominantly inherited disease in which most individuals are born with one defective allele encoding for either hamartin (TSC1) or tuberin (TSC2), with a somatic loss of the other allele leading to abnormal neurodevelopment and upregulation of cell growth in susceptible tissues. Ninety percent of affected individuals have brain involvement, including epilepsy, cognitive impairment, autism, and/or sleep disorders. In the stochastic, cerebral mouse model of Tsc1, loss of function of hamartin is induced in the CNS by injection of an adeno-associated virus (AAV) vector encoding Cre recombinase into the cerebral ventricles of homozygous Tsc1^flox/flox mice at birth. In the brain, Tsc1 loss leads to increased proliferation of subventricular zone cells, disrupted neuronal migration and cortical cytoarchitecture, dysmyelination, and microglia-mediated inflammation, ultimately resulting in early mortality. Systemic administration of an AAV9 vector encoding human hamartin at postnatal day 21 significantly ameliorated these abnormalities at 3 and 6 weeks post-injection and markedly extended survival in this TSC1 mouse model. This work reveals the ability of hamartin replacement therapy to reverse some of the brain abnormalities caused by its loss in different cell types and provides support for the potential use of gene replacement therapy in the treatment of TSC1 patients.

Tau is a primary target for immunotherapy in Alzheimer's disease (AD). Recent studies have shown the potential of anti-tau fragment antibodies in lowering pathological tau levels in vitro and in vivo. Here, we compared the effects of single-chain variable fragments (scFvs) derived from the well-characterized monoclonal antibodies PHF1 and MC1. We used adeno-associated virus 1 (AAV1) to deliver scFvs to skeletal muscle cells in 8-week-old P301S tau transgenic mice. We evaluated motor and behavioral functions at 16 and 23 weeks of age and measured misfolded, soluble, oligomeric, and insoluble brain tau species. Monotherapy with scFv-MC1 improved motor and behavioral functions more effectively than scFv-PHF1 or combination therapy. Brain glucose metabolism also benefited from scFv-MC1 treatment. Surprisingly, combining scFvs targeting early (MC1) and late (PHF1) tau modifications did not produce additive or synergistic effects. These results confirm that intramuscular AAV1-mediated scFv-MC1 gene therapy holds promise as a potential treatment for AD. Our findings also suggest that combining scFvs targeting different tau epitopes may not necessarily enhance efficacy if administered together in a prevention paradigm. Further research is needed to explore whether other antibodies' combinations and/or administration schedules could improve the efficacy of scFv-MC1 alone.

X-linked agammaglobulinemia (XLA) is a rare inborn error of immunity caused by loss-of-function mutations in the gene encoding Bruton's tyrosine kinase (BTK). XLA patients lack mature B cells and have negligible antibody levels, leaving them susceptible to recurrent bacterial and chronic viral infections. Autologous hematopoietic stem cell gene therapy with gene-corrected HSC may serve as a promising treatment of XLA; this therapy would provide a one-time cure and would replace lifelong immunoglobulin replacement therapy. Due to the requirement of strict physiological regulation of BTK gene expression, a site-specific editing strategy was designed to insert a BTK cDNA transgene directly into its endogenous locus. To study the effectiveness of this therapy, murine lineage-negative hematopoietic cells from a murine model of XLA were edited using CRISPR-Cas9/rAAV6 then transplanted into recipient XLA mice. Myeloablated XLA mice that received transplantation of Btk-corrected Lin- cells displayed high levels of engraftment, significant increases in their B cell levels, increased production of various immunoglobulins, improved B cell development in the bone marrow, increased B cell receptor diversity, and the ability to produce antigen-specific antibodies following immunization. Collectively, we have modeled a gene therapy strategy in a disease model of XLA and extensively validated the site-specific genome editing approach.

Plasminogen activator inhibitor-1 (PAI-1) deficiency is a rare disorder that causes moderate to severe bleeding and cardiac fibrosis, caused by mutation in the SERPINE-1 gene and no detectable circulating PAI-1 protein. There are currently no therapies that can effectively replace PAI-1 because the protein has a short half-life. An alternative approach to using recombinant protein is to endogenously increase circulating PAI-1 levels using mRNA therapy. Delivering mRNA encoding PAI-1 to the liver, a major site of PAI-1 synthesis, using lipid nanoparticles (mPAI-1) is a potential approach to increase circulating PAI-1 protein. Here, we developed mPAI-1, which induced expression of PAI-1 in vivo upon intravenous administration. In both wild-type (WT) mice and PAI-1 knockout mice, mPAI-1 induced supraphysiological circulating PAI-1 and inhibited fibrinolysis when measured ex vivo. In WT mice, plasma PAI-1 levels increased in a dose-dependent manner between 0.1 and 1 mg of mRNA per kg of body weight, peaking at 6 h post-injection and returning to baseline by 48 h. There was consistent production of PAI-1 after repeat dosing of mPAI-1 in the same mice. Expression of PAI-1 using mRNA-based approaches has the potential to be a preventive therapy for bleeding and cardiac fibrosis for PAI-1-deficient patients.

Optogenetics offers a minimally invasive, low-fatigue, and temporally precise alternative to electrical stimulation for skeletal muscle control. After opsin expression in muscle cells, contraction can be stimulated with light. Obstructive sleep apnea, characterized by repeated airway collapse during sleep, suits this approach, as upper airway muscles are readily accessible via the oral cavity, and require stimulation synchronized to respiration. This study compared wild-type (adeno-associated virus 9 [AAV9]) and engineered (AAVMYO) viral vectors for the delivery of identical muscle-specific optogenetic constructs in rats. Three weeks after intramuscular injections, both vectors produced comparable opsin expression in the tongue (p = 0.54) and near-zero expression in non-target tissues. AAVMYO-treated animals had greater light-evoked increases in muscle activation than those treated with AAV9 (8.5-fold vs. 2.0-fold; p < 0.0001). Conversely, AAV9-treated animals had greater light-evoked airway dilation (2.1 mm² vs. 0.3 mm²; p = 0.02). By 12 weeks, opsin expression declined to near-zero (vs. 3 weeks; p < 0.0001), light stimulation no longer increased muscle activation (p > 0.05), and anti-AAV antibodies had significantly increased (p < 0.0001). Unlike in mice, AAVMYO did not consistently outperform AAV9 in delivering gene therapy to rat muscles. Despite this, these data support optogenetics for obstructive sleep apnea, although sustained efficacy requires a better understanding of host immune responses and potentially transient immune suppression.

Recombinant adeno-associated viruses (rAAV) have emerged as a preferred strategy for in vivo gene delivery. However, the immune response to rAAV presents a major limitation, leading to serious adverse events in clinical trials. This study investigates the interaction between rAAV and the innate immune system. A whole blood assay (WBA) was used to assess complement activation and cytokine release upon stimulation with rAAV in 20 healthy blood donors. Results demonstrate that AAV2 and AAV8 capsids can activate the complement system, primarily through the antibody-dependent classical pathway. Complement activation also occurred in some of the seronegative donors showing the contribution of the alternative pathway. In this WBA setting, there were significant increases in the release of various cytokines and chemokines, with monocytes, natural killer cells, T cells, and B cells identified as the responding cell types using transcriptomics. Cytokine and/or chemokine release was more prominently observed with AAV2 compared with AAV8 and was enhanced by the presence of pre-existing anti-capsid antibodies. Interferon-α release appeared directly dependent on the cytosine-phosphate-guanine (CpG) content of the vector genome. These findings underscore the effects of innate and adaptive immunity to rAAV capsid and genome on the activation of complement pathways and release of inflammatory mediators.

Surfeit locus protein 1 (SURF1)-related Leigh syndrome is an early-onset neurodegenerative disorder characterized by a reduction in complex IV activity that disrupts mitochondrial function. Currently, there are no disease-modifying treatments available. Previously, we reported that a gene replacement therapy for SURF1-related Leigh syndrome was developed, which showed improved complex IV activity and restored exercise-induced lactate acidosis, as well as a high safety profile in wild-type (WT) mice. However, further investigations of this original SURF1 vector design uncovered cytotoxicity in multiple tissues of WT rats despite having minimal immune responses. We hypothesized that this cytotoxicity was elicited by SURF1 protein overexpression driven by a strong ubiquitous promoter, CBh. Here, we report the development of an improved gene therapy for SURF1 Leigh syndrome by utilizing a different promoter and polyadenylation sequence. Our data showed that, with lower SURF1 protein expression, the new design conferred a similar level of efficacy, but with minimal cytotoxicity in mice or rats. We propose this new vector design as a promising therapeutic candidate for SURF1-related Leigh syndrome, warranting further translational studies.