Molecular Therapy-Methods & Clinical Development最新文献

The aim of this research was to explore the therapeutic capabilities of extracellular vehicles (EVs) derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) that had been subjected to heat shock pretreatment, in treating psychiatric disorders induced by sleep deprivation in mice. The EVs were isolated and characterized, while Western blotting was utilized to assess the expression of exosomal markers and heat shock protein 70 (HSP70). To evaluate the impact of EV treatment on anxiety-like behavior and cognitive impairment in sleep-deprived (SD) mice, open field test, plus maze test and Y-maze task were conducted. Heat shock pretreatment significantly increased the expression of HSP70 in EVs. Administration of EVs from heat shock pretreated hUC-MSCs improved anxiety-like behavior and cognitive function in sleep-deprived mice. Furthermore, EV treatment promoted synaptic protein expression, HSP70 expression and inhibited neuroinflammation in the hippocampus of SD mice. Western blotting analysis also revealed that EV treatment reduced the levels of TLR4 and p65 in the hippocampus. EVs from heat shock-pretreated hUC-MSCs have therapeutic potential for sleep deprivation-induced psychiatric disorders by regulating neuroinflammation and synaptic function in mice.

Self-complementary AAV vectors (scAAV) employ a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. Yet, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These non-canonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors termed covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of employing a mutant ITR, we utilized a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vector. Treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to development of improved human gene therapy drugs.

The transgene toggling device is recognized as a powerful tool for gene and cell-based biological research and precision medicine. However, many of these devices often operate in binary mode, exhibit unacceptable leakiness, suffer from transgene silencing, show cytotoxicity, and have low potency. Here, we present a novel transgene switch, SIQ, wherein all the elements for gene toggling are packed into a single vector. SIQ has superior potency in inducing transgene expression in response to tebufenozide compared to the Gal4/UAS system, while completely avoiding transgene leakiness. Additionally, the ease and versatility of SIQ make it possible with a single construct to perform transient transfection, establish stable cell lines by targeting a predetermined genomic locus, and simultaneously produce adenovirus for transduction into cells and mammalian tissues. Furthermore, we integrated a cumate switch into SIQ, called SIQmate, to operate a Boolean AND logic gate, enabling swift toggling-off of the transgene after the removal of chemical inducers, tebufenozide and cumate. Both SIQ and SIQmate offer precise transgene toggling, making them adjustable for various researches, including synthetic biology, genome engineering, and therapeutics.

The hemophilias are the most common severe inherited bleeding disorders and are caused by deficiency of clotting factor (F) VIII (hemophilia A) or FIX (hemophilia B). The resultant bleeding predisposition significantly increases morbidity and mortality. The ability to improve the bleeding phenotype with modest increases in clotting factor levels has enabled the development and regulatory approval of adeno-associated viral (AAV) vector gene therapies for people with hemophilia A and B. The canine hemophilia model has proven to be one of the best predictors of therapeutic response in humans. Here, we report long-term follow-up of 12 companion dogs with severe hemophilia that were treated in a real-world setting with AAV gene therapy. Despite more baseline bleeding than in research dogs, companion dogs demonstrated a 94% decrease in bleeding rates and 61% improvement in quality-of-life over a median of 4.1 years (range 2.6-8.9). No new anti-transgene immune responses were detected; 1 dog with a preexisting anti-FVIII inhibitor achieved immune tolerance with gene therapy. Two dogs expressing 1-5% FVIII post gene therapy experienced fatal bleeding events. These data suggest AAV liver-directed gene therapy is efficacious in a real-world setting, but should target expression > 5% and closely monitor those with levels in the 1-5% range.

Despite the success of combination antiretroviral therapies (cART) in HIV treatment, a cure for HIV remains elusive. Scientists postulate that HIV latent reservoirs may be a vital target in curative strategies. Vorinostat is a latency-reversing agent which has demonstrated some effectiveness in reactivating latent HIV, but complementary therapies may be essential to enhance its efficacy. One such approach may utilize the CRISPR/Cas9 system which has evolved to include transcriptional activators such as dCas9-VPR. In this study, we explored the effects of combining vorinostat coupled with gesicle-mediated delivery of dCas9-VPR in promoting the transcription of integrated HIV proviruses in HIV-NanoLuc CHME-5 microglia and J-Lat 10.6 lymphocytes. We confirmed that dCas9-VPR ribonucleoprotein complexes can be packaged into gesicles and application to cells successfully induced HIV transcription through interactions with the HIV LTR. Vorinostat also induced significant increases in proviral transcription but generated inhibition of cellular proliferation (microglia) or cell viability (lymphocytes) starting at 1000nM and higher concentrations. Experiments combining dCas9-VPR gesicles and vorinostat confirmed the enhanced transcriptional activation of the HIV provirus in microglia but not lymphocytes. Thus, a combination of dCas9-VPR gesicles with other latency reversing agents may provide a complementary method to activate latent HIV in future studies utilizing patient-derived cells or small animal models.

Genetically engineered macrophages (GEMs) have emerged as an appealing strategy to treat cancers, but are largely impeded by the cell availability and technical challenges in gene transfer. Here, we develop an efficient approach to generate large-scale macrophages from human induced pluripotent stem cells (hiPSCs). Starting with 1 T150 dish of 10⁶ hiPSCs, more than 10⁹ mature macrophages (iMacs) could be generated within 1 month. The generated iMacs exhibit typical macrophage properties such as phagocytosis and polarization. We then generate hiPSCs integrated with an IL12 expression cassette in AAVS1 locus to produce iMacs secreting IL12, a strong pro-immunity cytokine. hiPSCs derived iMacs_IL12 prevent cytotoxic T cell exhaustion and activate T cells to kill different cancer cells. Furthermore, iMACs_IL12 display strong anti-tumor effects in a T cell dependent manner in subcutaneously or systemically xenografted mice of human lung cancer. Therefore, we provide an off-the-shelf strategy to produce large-scale GEMs for cancer therapy.

Alpha-1 antitrypsin deficiency (AATD) is characterized by both chronic lung disease due to loss of wild-type AAT (M-AAT) antiprotease function and liver disease due to toxicity from delayed secretion, polymerization, and aggregation of misfolded mutant AAT (Z-AAT). The ideal gene therapy for AATD should therefore comprise both endogenous Z-AAT suppression and M-AAT overexpression. We designed a dual-function rAAV3B (df-rAAV3B) construct, which was effective at transducing hepatocytes, resulting in a considerable decrease of Z-AAT levels and safe M-AAT augmentation in mice. We optimized df-rAAV3B and created two variants, AAV3B-E12 and AAV3B-G3, to simultaneously enhance the concentration of M-AAT in the bloodstream to therapeutic levels and silence endogenous AAT liver expression in cynomolgus monkey. Our results demonstrate that AAV3b-WT, AAV3B-E12, and AAV3B-G3 were able to transduce the monkey livers and achieve high M-AAT serum levels efficiently and safely. In this non-deficient model, we did not find down-regulation of endogenous AAT. However, the dual-function vector did serve as a potentially “liver-sparing” alternative for high dose liver-mediated AAT gene replacement in the context of underlying liver disease.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disease caused by iduronate-2-sulfatase (IDS) deficiency, leading to accumulation of glycosaminoglycans (GAGs) and the emergence of progressive disease. Enzyme replacement therapy is the only currently approved treatment, but leaves neurological disease unaddressed. CSF-directed administration of AAV9.CB7.hIDS (RGX-121) is an alternative treatment strategy, but it is unknown if this approach will affect both neurologic and systemic manifestations. We compared the effectiveness of intrathecal (IT) and intravenous (IV) routes of administration (ROA) at a range of vector doses in a mouse model of MPS II. While lower doses were completely ineffective, a total dose of 1x10⁹gc resulted in appreciable IDS activity levels in plasma, but not tissues. Total doses of 1x10¹⁰gc and 1x10¹¹gc by either ROA resulted in supraphysiological plasma IDS activity, substantial IDS activity levels and GAG reduction in nearly all tissues and normalized zygomatic arch diameter. In the brain, a dose of 1x10¹¹gc IT achieved the highest IDS activity levels, greatest reduction in GAG content, and prevented neurocognitive deficiency. We conclude that a dose of 1x10¹⁰gc normalized metabolic and skeletal outcomes, while neurologic improvement required a dose of 1x10¹¹gc, thereby suggesting the prospect of a similar direct benefit in humans.

Patients with preexisting immunity to adeno-associated virus (AAV) are currently unable to receive systemic gene transfer therapies. This nonhuman primate study investigated the impact of immunosuppression strategies on gene transfer therapy safety/efficacy and analyzed plasmapheresis as a potential pretreatment for circumvention of pre-existing immunity or redosing. In Part 1, animals received delandistrogene moxeparvovec (SRP-9001), an AAVrh74-based gene transfer therapy for Duchenne muscular dystrophy. Cohort 1 (control, n=2) received no immunosuppression; cohorts 2-4 (n=3/cohort) received prednisone at different time points; and cohort 5 (n=3) received rituximab, sirolimus, and prednisone before and after dosing. In Part 2, cohorts 2-4 underwent plasmapheresis before redosing; cohort 5 was redosed without plasmapheresis. We analyzed safety, immune response (humoral and cell-mediated responses and complement activation), and vector genome distribution. After 2-3 plasmapheresis exchanges, circulating anti-AAVrh74 antibodies were reduced, and animals were redosed. Plasmapheresis was well tolerated, with no abnormal clinical or immunological observations. Cohort 5 (redosed with high anti-AAVrh74 antibody titers) had hypersensitivity reactions, which were controlled with treatment. These findings suggest that plasmapheresis is a safe and effective method to reduce anti-AAV antibody levels in nonhuman primates prior to gene transfer therapy. The results may inform human studies involving redosing or circumvention of preexisting immunity.

The transfection efficiency and stability of the delivery vehicles of plasmid DNA (pDNA) are critical metrics to ensure high-quality and high-yield production of viral vectors. We previously identified that the optimal size of pDNA/poly(ethylenimine) (PEI) transfection particles is 400 to 500 nm and developed a bottom-up assembly method to construct stable 400-nm pDNA/PEI particles and benchmarked their transfection efficiency in producing lentiviral vectors (LVVs). Here, we report scale-up production protocols for such transfection particles. Using a 2-inlet confined impinging jet (CIJ) mixer with a dual syringe pump setup, we produced a 1-L batch at a flow rate of 100 mL/min; and further scaled up this process with a larger CIJ mixer and a dual peristaltic pump array, allowing for continuous production at a flow rate of 1 L/min without a lot-size limit. We demonstrated the scalability of this process with a 5-L lot and validated the quality of these 400-nm transfection particles against the target product profile, including physical properties, shelf and on-bench stability, transfection efficiency, and LVV production yield in both 15-mL bench culture and 2-L bioreactor runs. These results confirm the potential of this particle assembly process as a scalable manufacturing platform for viral vector production.