Molecular Therapy-Methods & Clinical Development最新文献

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.

RNA-targeting CRISPR-Cas systems have emerged as alternatives to RNA-interference technology to knock down specific RNA transcripts. In particular, Cas13d derived from Ruminococcus flavefaciens (CasRx, RfxCas13d) has generated interest due to its superior knockdown efficiencies; however, accumulating evidence indicates that CasRx is prone to inducing transcriptome alterations due to its tendency to cleave bystander RNAs. High-fidelity Cas13d (hfCas13d) derived from CasRx and DjCas13d, an ortholog of Cas13d derived from Ruminococcus sp. UBA7013 (gut metagenome), are two recently identified variants that are superior to CasRx, as they both show a reduced tendency to cleave bystander RNAs. In this study, we created a resource of adeno-associated viral (AAV) vectors designed to deliver Cas13d, including hfCas13d and DjCas13d. We directly compared hfCas13d and DjCas13d for their on- and off-target potential in 293FT and neuro 2A cells. Specifically, we examined their ability to knockdown several endogenous and ectopically expressed transcripts using several different guide RNAs (gRNAs), and we examined knockdown specificity using a combination of reporter assays, RNA integrity analysis, and RNA sequencing (RNA-seq). We report that while both of these enzymes exhibit generally similar levels of knockdown potential, with DjCas13d sometimes outperforming hfCas13d, hfCas13d consistently caused significantly fewer transcriptome alterations when targeting highly expressed genes compared to DjCas13d.

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.

This report describes the distribution and transgene expression of two non-cytotoxic, replication-defective (rd) herpes simplex virus (HSV) vectors, JΔNI7 and JΔNI8, following intraperitoneal delivery to newborn mice. The two vectors are functionally defective for all immediate-early genes, and JΔNI8 is further deleted for the UL41 endonuclease (vhs). Both vectors were engineered to express a red luciferase gene from the LAT locus to track vector distribution and gene expression in vivo. A comparison of reporter gene activities under the control of four different promoters in JΔNI7 showed that the strongest expression was achieved with the CAG promoter. Distribution analysis at 1 week post-injection showed transgene expression in multiple tissues, but at 4 weeks, high-level expression was limited to the spinal cord, skin, and muscles. JΔNI8 showed rapid clearance of vector DNA in most tissues, suggesting a role for the vhs gene in vector stability. Compared with wild-type KOS strain injections, JΔNI7-based non-cytotoxic rdHSV did not induce substantial CD45⁺ immune-cell infiltration or tissue destruction, suggesting that our rdHSV vectors are safe. Taken together, these results demonstrate tissue-specific, durable transgene expression following systemic delivery of rdHSV vectors, suggesting their potential for systemic gene therapy for newborns with skin or neuromuscular diseases.

Testing for binding or neutralizing antibodies (NAbs) to adeno-associated virus (AAV) is part of the laboratory assessment of people with hemophilia considering AAV-based gene therapy. We evaluated the natural history of NAb titers to AAV serotype 5 (AAV5) in adult males ≥18 years old with hemophilia B (factor IX ≤ 2%) during the lead-in period of a phase 3 trial prior to the infusion of etranacogene dezaparvovec to characterize NAb in addition to immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-AAV5 binding antibody changes over time. At screening, 48% (32/67) of enrolled participants had detectable NAbs (NAb+) with a median titer of 58 (range: 9-3,440). Participant-specific lead-in periods differed and included discontinuers (median duration: 240 days; range: 1-360). The median intra-participant coefficient of variation of NAb titer over time was 25% (range: 2%-154%). NAb seropositivity was associated with older age (p = 0.0065). For participants with detectable anti-AAV5 NAbs and IgG, there was a high correlation of titers at each visit (median r = 0.96; range: 0.92-0.99). IgM anti-AAV5 antibodies were detectable in only 9% of participants, and seroconversion was infrequent. In conclusion, AAV5 NAb test results were consistent over 6 months, which informs the timing of NAb screening when considering gene therapy for hemophilia B.