Molecular Therapy-Methods & Clinical Development最新文献

This study evaluated the efficacy and safety of adeno-associated virus serotype 8 (AAV8)-aflibercept for treating choroidal neovascularization (CNV) in mice model, utilizing single-cell RNA sequencing (scRNA-seq) to investigate retinal effects. AAV8-aflibercept was administered via subretinal injection to mice, followed by laser-induced CNV modeling. Successful expression was achieved following subretinal injection of AAV8-aflibercept, aflibercept protein expression showed a gradual decline after peaking at week 4, but remained at relatively stable levels through weeks 8-24. The therapeutic effect was assessed using fluorescein angiography and quantified. AAV8-aflibercept significantly decreased CNV areas across dosages from 3 × 10⁷ to 1 × 10⁹ GC/eye, with minor retinal thinning and retinal pigment epithelium (RPE) alterations observed only at dosages exceeding 1 × 10⁹ GC/eye. Twelve distinct cell types were identified by single-cell RNA sequencing (scRNA-seq). RPE cells were the primary target, with 29.7% expressing the transduced gene. Target gene expression in RPE cells mainly involved RNA splicing and mRNA processing, with no strong activation signatures were observed in inflammation, apoptosis, or autophagy pathways. Upregulation of Gadd45b, Lrrc2, Lcn2 was noted in the AAV8-aflibercept group, suggesting a stress response. AAV8-aflibercept demonstrated significant efficacy and safety in treating CNV, with scRNA-seq providing insights into cellular changes.

Autosomal dominant mutations in ELANE (elastase, neutrophil expressed) cause severe congenital neutropenia (CN) and cyclic neutropenia (CyN). Inhibiting ELANE expression, either by CRISPR-Cas9-mediated ELANE knockout or promoter targeting using CRISPR-Cas9 nickase, has emerged as a promising gene therapy strategy to restore defective granulocytic differentiation of transplantable hematopoietic stem cells from CN patients. We developed an adenine base editor (ABE)-mediated approach targeting two nucleotides in the ELANE promoter to suppress neutrophil elastase expression, called PRECISE. Analysis of mRNA- and protein-based delivery of ABE revealed that although both platforms were effective in editing hematopoietic stem and progenitor cells from healthy donors with over 80% editing, only protein-based ABE delivery achieved over 68% editing in CN patient cells. Interestingly, 10%-19% editing in CN patients' hematopoietic cells using ABE mRNA restored their granulocytic differentiation in vitro, with a marked expansion and differentiation of ABE ribonucleoprotein (RNP)-edited cells. PRECISE-edited neutrophils retained normal function, including neutrophil extracellular trap formation, oxidative burst, and phagocytosis. Genome integrity analysis showed no genomic alterations or chromosomal aberrations, and only two off-target edits confined to non-coding intronic regions. In conclusion, PRECISE represents a translationally relevant base-editing strategy for ELANE-associated CN and CyN that addresses ELANE mutation heterogeneity.

Wet age-related macular degeneration (wAMD) is a leading cause of vision loss and is characterized by choroidal neovascularization (CNV). Current CNV management requires multiple treatments and lacks long-term efficiency, creating a need for better therapeutics. wAMD pathogenesis is associated with excessive activation of the complement system, contributing to retinal damage. Therefore, we generated a vector expressing the small alternative pathway-targeting nanobody, hC3Nb1, to treat wAMD. We demonstrate that hC3Nb1 is efficiently expressed and secreted by mammalian cells and shows full alternative pathway and partial classical pathway inhibition in vitro. A dual-promoter approach was used to generate a lentiviral-based vector for co-expression of hC3Nb1 and marker protein eGFP. Profound and safe hC3Nb1-expression, along with its secretion from the retinal pigment epithelium (RPE), was confirmed following subretinal injection of nanobody expressing-vector in mice. The therapeutic potential of vector-encoded hC3Nb1 was demonstrated in vitro by protecting RPE from complement-mediated stress, and in vivo by reducing laser-induced CNV sizes in a mouse model consistent with complement inhibition. For the first time, nanobodies expressed in the eye are used therapeutically, and our findings suggest that hC3Nb1-based gene therapy may be a safe and long-acting treatment for wAMD and other chorioretinal diseases with dysregulated complement activation.

Mutations in the myocilin gene (MYOC) are the leading genetic cause of primary open angle glaucoma (POAG), the most common glaucoma type. These mutations trigger a toxic gain-of-function phenotype, causing the misfolded MYOC protein to accumulate in the endoplasmic reticulum (ER) of trabecular meshwork (TM) cells, leading to ER stress, TM cell death, and elevation of intraocular pressure (IOP). Here, we demonstrate that the delivery of Cas9 mRNA via a cationic lipid polymer (lipoplex) targets the TM selectively and edits the MYOC gene, thereby rescuing a mouse model of glaucoma. A single intracameral injection of Cre-mRNA lipoplex resulted in mutant MYOC expression in the TM, leading to glaucoma in a recently developed Cre-inducible mouse model of glaucoma. Lipoplex encapsulating Cas9 and guide RNA targeting MYOC knocked out MYOC, reduced intracellular accumulation of mutant MYOC, and relieved ER stress, thereby rescuing a mouse model of MYOC-associated glaucoma. Our studies further establish the ocular safety and efficacy of non-viral Cas9-sgRNA delivery to the TM, offering a novel, one-time therapeutic strategy for inherited glaucoma caused by MYOC mutations.

Gene delivery to critical cell types within the kidney can enable preclinical evaluation of gene therapies for kidney disease. The novel adeno-associated virus AAV.cc47 was discovered after sequential evolution in mice, pigs, and macaques and improved transduction in multiple tissues but without in-depth exploration of the kidney. We observed robust kidney transduction by AAV.cc47 vectors in mice in vivo and in human kidney organoids compared to AAV9, mostly within the proximal tubule (PT) epithelium. We then developed a quantitative analysis method of transgene expression utilizing automated classification of nephron cell types coupled with cellular expression. Despite exhibiting similar biodistribution to AAV9 in renal and extrarenal tissues, AAV.cc47 consistently transduced the kidney at higher efficiency, with >80% of PT epithelium transduced at low, systemically administered vector dose. Self-complementary AAV.cc47 vectors appear to transduce a subset of PT epithelium, with undetectable transduction of non-PT cells. This method could be adapted to evaluate different AAV vectors transducing other kidney cell types. We also demonstrate the utility of dual AAV.cc47 vectors to increase genome payload capacity for kidney gene transfer. AAV.cc47 represents a promising vector for use as a research tool and possibly clinical application for kidney disease.

Charge detection mass spectrometry (CD-MS) has previously been shown to be effective for analyzing predominantly clean bulk drug substances and drug products of recombinant adeno-associated virus (rAAV). CD-MS holds potential for assessing and monitoring the quality attributes of rAAV during the manufacturing process, from harvest to drug product. The mass range of rAAV material has been well characterized experimentally by CD-MS to agree with theoretical values for protein and target genome masses. CD-MS measures the charge range derived from native electrospray ionization of rAAV materials. Ions of rAAV can be separated in data processing using both mass and charge characteristics to improve certainty of rAAV analytics. We demonstrate the capabilities of CD-MS analysis for examining in-process samples from the bulk harvest, affinity chromatography, and anion exchange chromatography. Our results show that the harvest material maintains consistent empty/partial/full ratios when compared to the affinity purified sample. Process impurities were detected for each stage of production and compared to current industry standards. We find that CD-MS is highly reproducible and can be used to effectively track the production process from harvest to bulk drug substance, providing quantitative results at each stage.

The variability in structure and stability of adeno-associated virus serotype 9 (AAV9) in response to changes in buffer composition, pH, and temperature was investigated using charge detection mass spectrometry (CDMS). AAV9 virus-like particles (VLPs) consisting of only viral protein (VP) 3 and wild-type AAV9 capsids (i.e., capsids containing varying stoichiometries of VP1, VP2, and VP3) showed differences in structure, indicating that these different VP stoichiometries and compositions may contribute substantially to conformational heterogeneity. Significant differences in AAV9 structure and stability were observed in ammonium acetate (AA) vs. phosphate buffered saline (PBS) solutions under some conditions. At 37°C under acidic conditions, AAV capsids fell apart in AA, whereas in PBS, capsids underwent structural compaction. Subsequent nuclease binding experiments indicated that partially extruded DNA was the likely origin of this structural compaction that occurred under different physical and chemical conditions. Results from one freeze-thaw cycle indicated that the capsids degraded by a similar mechanism to that in acidified solution. The structural complexity revealed by CDMS highlights the advantages of this biophysical characterization method in providing, for the first time, a holistic insight into the potential heterogeneous conformational transitions of AAV9 during purification, storage, and the natural infection process.

Antibody-based immunotherapies are promising; however, their application remains limited to acute diseases due to rapid clearance of antibodies in vivo. Some chronic conditions could benefit from sustained therapeutic antibody expression. One such instance is human immunodeficiency virus type 1 (HIV-1), where the efficiency of broadly neutralizing antibodies by passive immunization has been limited in clinical trials. B cell editing to enable sustained production of an antibody of interest in vivo could address this issue. However, the long-term potential of this approach and feasibility to perform editing in B cells from people living with HIV remain to be determined. We investigated editing of rhesus macaque B cells from healthy or simian/human immunodeficiency virus (SHIV)-infected animals to model this approach. An antibody-encoding cassette was inserted in the immunoglobulin locus by CRIPSR-Cas9-mediated ex vivo B cell editing. Similar indel efficiencies were achieved in B cells from both uninfected and infected animals, and expression of the antibody of interest was detected in up to 10% of uninfected B cells. This study paves the way for future in vivo work to assess the long-term potential of this approach and its impact on B cell development and function in an immunocompetent in vivo nonhuman primate model of HIV persistence and cure.

Recombinant AAV (rAAV) vectors are a leading viral vector for gene therapy. Viral genome (Vg) titer is the primary method to determine potency of rAAV and dosing in preclinical/clinical studies. However, the rAAV genome comprises a heterogeneous population. These particles not only contain the intact genome but also include numerous truncated species, which likely lack functionality and may induce adverse effects. Consequently, the Vg titer does not accurately reflect the integrity of the rAAV genome. Currently, there is no reliable quantitative method available. In this study, we demonstrate that there is a disconnect between Vg titer and the activity of rAAV by using multiple vectors and high-throughput imaging assays. Importantly, we have developed a novel, high-throughput RNA-DNA hybrid capture-multiplex meso scale discovery (MSD) method for characterizing the integrity of the rAAV genome. This method quantifies the intact versus truncated genomes of both the plus and minus strands individually with high sensitivity and specificity. The integrity data generated by our novel method exhibits a strong correlation with the activity of the rAAV. We anticipate that our new method will significantly improve preclinical/clinical studies, enhance vector design, and increase delivery efficiency. Furthermore, this method can be used to characterize and quantitate RNA and DNA in various fields.

Recombinant adeno-associated virus (rAAV) vectors are widely used in gene therapy due to their ability to transduce various cell types and tissues, sustained gene expression, and relatively safe profile. However, the production of rAAV vectors using the baculovirus/Sf9 system can result in the mispackaging of the baculoviral (recombinant baculoviral [rBV]) DNA. This study aims to characterize and quantify these rBV DNA impurities in rAAV products. We developed a multiplex digital PCR method to model, characterize, and accurately determine rBV DNA impurities. Our findings indicate that rBV DNA within 5 kb of the inverted terminal repeat (ITR) and the mini-F region has a higher probability of being mispackaged into rAAV particles than other regions. Additionally, using regular PCR plus agarose gel analysis and digital PCR, full-length kanamycin and gentamicin antibiotic genes were detected and quantitated in the rAAV. The study also revealed a strand-selective mispackaging of rBV DNA, with no correlation between the amount of rBV DNA impurity and the vector's size conflicting with the prevalent belief that smaller vectors will contain more rBV impurities. These results provide insights into the mechanisms of rBV DNA impurity formation and suggest strategies to reduce these impurities, thereby enhancing the safety and efficacy of rAAV-based gene therapies.