Molecular Therapy-Methods & Clinical Development最新文献

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.

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.

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.

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.

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.

Development of oligonucleotide-based therapeutics has been limited by the lack of effective in vivo delivery vehicles. We previously showed that the presence of an H-type excitonic dimer formed by the covalent binding of two fluorophores with significant transition dipoles on opposite ends of peptide or nucleic acid sequences of interest facilitated their delivery into live cells in vitro. Here, we evaluated delivery of a fluorogenic anti-sense oligonucleotide (ASO) complementary to β-actin (ACTB) mRNA into human leukocytes in vivo using a humanized mouse model. We observed delivery of the ASO into human leukocytes at a median of ≥94% in blood, spleen, bone marrow, and liver with half-lives of at least 72 h. Additionally, we detected the ASO in a significant proportion of human leukocytes within difficult-to-penetrate tissues such as brain and gut. The ASO localized to the perinuclear space within target cells and mediated reductions in bone marrow ACTB transcripts after a single intravenous injection. The delivery system described herein is a technology platform with the capacity for highly efficient systemic delivery of therapeutic oligonucleotides that will facilitate development of a new class of drugs for treatment of HIV and other pathologic conditions.

Initial entrapment of nucleic acids in lipid nanoparticles (LNPs) is dependent on the use of ionizable cationic lipids, which draw nucleic acids into lipid particles at low pH in the presence of ethanol. Manufacturing of fully formed LNPs is completed by buffer exchange and removal of ethanol. We studied particle morphology at intermediate pH values during buffer exchange using an fluorescence resonance energy transfer (FRET) assay to indicate particle interactions, particle sizing, cryo-electron microscopy (cryo-EM), and small-angle X-ray scattering (SAXS). We compared LNPs formed by different ionizable lipids, including DLin-MC3-DMA, ALC-0315, and SM-102, used, respectively, in the Food and Drug Administration (FDA)-approved products, Onpattro, Comirnaty, and Spikevax. FRET and cryo-EM studies confirmed that particle interaction and fusion occurred during buffer exchange. By dialyzing LNPs in various buffers, we found that stable particles with bleb-like protrusions were formed at intermediate pH (i.e., pH 5.5). Fusion and particle growth occurred at higher pH values for SM-102 LNPs, reflecting the higher pKa of SM-102. SAXS profiles showed that, when fully formed at the final pH of 7.4, MC3 and ALC-0315 LNPs had lost bilayer-like structures, which were present after particle formation at pH 4. In contrast, LNPs produced with 1,2-dioleyloxy-3-dimethylaminopropane (DODMA) and SM-102 retained some bilayer-like structures at pH 7.4.

[This corrects the article DOI: 10.1016/j.omtm.2023.101154.].