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.

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.

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.].

Persistent antigen stimulation and inflammatory environments drive exhaustion, senescence, and activation-induced cell death, impairing both endogenous and therapeutic T cells. Understanding the mechanisms underlying T cell dysfunction is critical for improving immunotherapies. While the transcription factor forkhead box protein P3 (FOXP3) is primarily known for its role in regulatory T cell development and maintenance, recent studies suggest it may also influence effector T cell function. However, its impact on therapeutic T cells, including CAR T cells, remains poorly defined. Here, we used non-viral CRISPR-Cas9 editing to knockout FOXP3 in CD19-directed CAR T cell products (TCPs) generated via lentiviral transduction. FOXP3 expression was upregulated at both the protein and RNA level following CAR stimulation. Compared to unmodified CAR TCPs, FOXP3-KO CAR TCPs showed comparable exhaustion profiles but enhanced cytokine production and prolonged cytotoxic function across repeated antigen challenges. These findings identify FOXP3 as a context-dependent modulator of CAR T cell function and suggest that its disruption may enhance therapeutic potency without exacerbating exhaustion. FOXP3 targeting may represent a complementary strategy to improve the functional resilience of CAR T cell therapies in cancer or autoimmune disease.

Cell substrate utilized in the production of biologics intended for human use need to be cleared from mycoplasma contaminant as described in pharmacopoeia. While the gold-standard method for mycoplasma detection involves lengthy microbiology and cell-based assays, nucleic acid technologies offer the possibility to accelerate testing. In this study, we develop a qPCR assay capable of specifically detecting 11 mycoplasma species relevant to pharmacopoeias with only two primers and two hydrolysis probes, which greatly facilitates operations. While the primers cross-react with bacterial species, the specificity conferred by the hydrolysis probes allows for confident interpretation of the qPCR result and unambiguous statement regarding the presence of mycoplasma in the test article. Amplicon sequencing can further confirm the identity of contaminants. This comprehensive assay can therefore be of great help to quality control laboratories embedded in biologics production sites, which need to provide results in a timely manner and contribute to root cause analysis in case of contamination.