Optical Control of Mononegavirus Gene Expression and Replication.

Mononegaviruses are promising tools as oncolytic and transgene vectors for gene therapy and regenerative medicine. However, when mononegaviruses are used for therapeutic applications, the viral activity must be strictly controlled due to concerns about toxicity and severe side effects. With this technology, mononegavirus vectors can be grown where they are intended and can be easily removed when they are no longer needed. In particular, a photoswitch protein called Magnet (consisting of two magnet domains) is incorporated into the hinge region between the connector and methyltransferase domains of the mononegavirus polymerase protein (L protein) to disrupt the L protein functions. Blue light (470 ± 20 nm) irradiation causes the dimerization of the two magnet domains, and the L protein is restored to activity, allowing viral gene expression and virus replication. Since the magnet domains' dimerization is reversible, viral gene expression and replication cease when blue light irradiation is stopped.

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Lentivirus-mediated gene therapy corrects ribosomal biogenesis and shows promise for Diamond Blackfan anemia.

This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.

Angiogenic Gene Therapy for Refractory Angina: Results of the EXACT Phase 2 Trial.

BACKGROUND: XC001 is a novel adenoviral-5 vector designed to express multiple isoforms of VEGF (vascular endothelial growth factor) and more safely and potently induce angiogenesis. The EXACT trial (Epicardial Delivery of XC001 Gene Therapy for Refractory Angina Coronary Treatment) assessed the safety and preliminary efficacy of XC001 in patients with no option refractory angina. METHODS: In this single-arm, multicenter, open-label trial, 32 patients with no option refractory angina received a single treatment of XC001 (1×1011 viral particles) via transepicardial delivery. RESULTS: There were no severe adverse events attributed to the study drug. Twenty expected severe adverse events in 13 patients were related to the surgical procedure. Total exercise duration increased from a mean±SD of 359.9±105.55 seconds at baseline to 448.2±168.45 (3 months), 449.2±175.9 (6 months), and 477.6±174.7 (12 months; +88.3 [95% CI, 37.1-139.5], +84.5 [95% CI, 34.1-134.9], and +115.5 [95% CI, 59.1-171.9]). Total myocardial perfusion deficit on positron emission tomography imaging decreased by 10.2% (95% CI, -3.1% to 23.5%), 14.3% (95% CI, 2.8%-25.7%), and 10.2% (95% CI, -0.8% to -21.2%). Angina frequency decreased from a mean±SD 12.2±12.5 episodes to 5.2±7.2 (3 months), 5.1±7.8 (6 months), and 2.7±4.8 (12 months), with an average decrease of 7.7 (95% CI, 4.1-11.3), 6.6 (95% CI, 3.5-9.7), and 8.8 (4.6-13.0) episodes at 3, 6, and 12 months. Angina class improved in 81% of participants at 6 months. CONCLUSIONS: XC001 administered via transepicardial delivery is safe and generally well tolerated. Exploratory improvements in total exercise duration, ischemic burden, and subjective measures support a biologic effect sustained to 12 months, warranting further investigation. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04125732.

The paths toward non-viral CAR-T cell manufacturing: A comprehensive review of state-of-the-art methods.

Although CAR-T cell therapy has emerged as a game-changer in cancer immunotherapy several bottlenecks limit its widespread use as a front-line therapy. Current protocols for the production of CAR-T cells rely mainly on the use of lentiviral/retroviral vectors. Nevertheless, according to the safety concerns around the use of viral vectors, there are several regulatory hurdles to their clinical use. Large-scale production of viral vectors under "Current Good Manufacturing Practice" (cGMP) involves rigorous quality control assessments and regulatory requirements that impose exorbitant costs on suppliers and as a result, lead to a significant increase in the cost of treatment. Pursuing an efficient non-viral method for genetic modification of immune cells is a hot topic in cell-based gene therapy. This study aims to investigate the current state-of-the-art in non-viral methods of CAR-T cell manufacturing. In the first part of this study, after reviewing the advantages and disadvantages of the clinical use of viral vectors, different non-viral vectors and the path of their clinical translation are discussed. These vectors include transposons (sleeping beauty, piggyBac, Tol2, and Tc Buster), programmable nucleases (ZFNs, TALENs, and CRISPR/Cas9), mRNA, plasmids, minicircles, and nanoplasmids. Afterward, various methods for efficient delivery of non-viral vectors into the cells are reviewed.

Progranulin AAV gene therapy for frontotemporal dementia: translational studies and phase 1/2 trial interim results.

GRN mutations cause progranulin haploinsufficiency, which eventually leads to frontotemporal dementia (FTD-GRN). PR006 is an investigational gene therapy delivering the granulin gene (GRN) using an adeno-associated virus serotype 9 (AAV9) vector. In non-clinical studies, PR006 transduced neurons derived from induced pluripotent stem cells of patients with FTD-GRN, resulted in progranulin expression and improvement of lipofuscin, lysosomal and neuroinflammation pathologies in Grn-knockout mice, and was well tolerated except for minimal, asymptomatic dorsal root ganglionopathy in non-human primates. We initiated a first-in-human phase 1/2 open-label trial. Here we report results of a pre-specified interim analysis triggered with the last treated patient of the low-dose cohort (n = 6) reaching the 12-month follow-up timepoint. We also include preliminary data from the mid-dose cohort (n = 7). Primary endpoints were safety, immunogenicity and change in progranulin levels in cerebrospinal fluid (CSF) and blood. Secondary endpoints were Clinical Dementia Rating (CDR) plus National Alzheimer's Disease Coordinating Center (NACC) Frontotemporal Lobar Degeneration (FTLD) rating scale and levels of neurofilament light chain (NfL). One-time administration of PR006 into the cisterna magna was generally safe and well tolerated. All patients developed treatment-emergent anti-AAV9 antibodies in the CSF, but none developed anti-progranulin antibodies. CSF pleocytosis was the most common PR006-related adverse event. Twelve serious adverse events occurred, mostly unrelated to PR006. Deep vein thrombosis developed in three patients. There was one death (unrelated) occurring 18 months after treatment. CSF progranulin increased after PR006 treatment in all patients; blood progranulin increased in most patients but only transiently. NfL levels transiently increased after PR006 treatment, likely reflecting dorsal root ganglia toxicity. Progression rates, based on the CDR scale, were within the broad ranges reported for patients with FTD. These data provide preliminary insights into the safety and bioactivity of PR006. Longer follow-up and additional studies are needed to confirm the safety and potential efficacy of PR006. ClinicalTrials.gov identifier: NCT04408625 .

Dual­regulated oncolytic adenovirus carrying ERCC1­siRNA gene possesses potent antitumor effect on ovarian cancer cells.

Ovarian cancer is a multifactorial and deadly disease. Despite significant advancements in ovarian cancer therapy, its incidence is on the rise and the molecular mechanisms underlying ovarian cancer invasiveness, metastasis and drug resistance remain largely elusive, resulting in poor prognosis. Oncolytic viruses armed with therapeutic transgenes of interest offer an attractive alternative to chemical drugs, which often face innate and acquired drug resistance. The present study constructed a novel oncolytic adenovirus carrying ERCC1 short interfering (si)RNA, regulated by hTERT and HIF promoters, termed Ad­siERCC1. The findings demonstrated that this oncolytic adenovirus effectively inhibits the proliferation, migration and invasion of ovarian cancer cells. Furthermore, the downregulation of ERCC1 expression by siRNA ameliorates drug resistance to cisplatin (DDP) chemotherapy. It was found that Ad­siERCC1 blocks the cell cycle in the G1 phase and enhances apoptosis through the PI3K/AKT­caspase­3 signaling pathways in SKOV3 cells. The results of the present study highlighted the critical effect of oncolytic virus Ad­siERCC1 in inhibiting the survival of ovarian cancer cells and increasing chemotherapy sensitivity to DDP. These findings underscore the potent antitumor effect of Ad­siERCC1 on ovarian cancers in vivo.

SARS-CoV-2 spike protein expression as an identification in quality control testing for Adenovector based COVID-19 vaccine.

AIM: Quality control testing of the vaccine for lot release is of paramount importance in public health. A recent pandemic caused by the SARS-CoV-2 virus brought together all spheres of vaccine to combat the virus. The scientific advancement in the development of vaccines facilitated the scientists to develop the vaccine against SARS-CoV-2 in a record time. Thus, these vaccines should be stringently monitored for their safety and efficacy as per the latest WHO and national regulatory guidelines, and quality control evaluation of the product should be done at national control laboratories before releasing the product into the market as it assures the quality and safety of the vaccine. METHODS: The SARS-CoV-2 exploited the ACE2 (Angiotensin Converting Enzyme 2) receptor, a surface protein on mammalian cells to gain entry into the host cells. The viral surface protein that interacted with the ACE2 receptor is the Spike protein of SARS-CoV-2. Thus, in the development of the vaccine and assessing its quality, the Spike protein of SARS-CoV-2 became an attractive immunodominant antigen. In National Institute of Biologicals, an apex body in the testing of biologicals in India, received the Adenovector (Adenovirus + vector) based COVID-19 vaccine, a finished product for quality evaluation. Due to the lack of a pharmacopeial monograph, the testing of the vaccine was done as per the manufacturer's specifications and methods. The routine assays of identification employed by the manufacturer do not reflect the expression of Spike protein which is required for the immune system to get activated. In this report, we showed the determination of Spike protein expression by immunoblotting and immunofluorescence for identification parameters in the quality testing of the COVID-19 vaccine. We determined the translation of the SARS-CoV-2 Spike gene cloned into an Adenovector. RESULTS: The results from these experiments indicated the expression of Spike protein upon infection of mammalian cells with viral particles suggested that the expression of immunodominant Spike protein of SARS-CoV-2 may be employed by quality control laboratories as a parameter for identification. CONCLUSION: The study suggested that the determination of the expression of Spike protein is pertinent to identifying the Adenovector based vaccines against COVID-19.

AAV mediated genome engineering with a bypass coagulation factor alleviates the bleeding phenotype in a murine model of hemophilia B.

It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20-42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice.

Development of an Improved Adenovirus Vector and Its Application to the Treatment of Lifestyle-Related Diseases.

The number of patients with lifestyle-related diseases such as type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has continued to increase worldwide. Therefore, development of innovative therapeutic methods targeting lifestyle-related diseases is required. Gene therapy has attracted considerable attention as an advanced medical treatment. Safe and high-performance vectors are essential for the practical application of gene therapy. Replication-incompetent adenovirus (Ad) vectors are widely used in clinical gene therapy and basic research. Here, we developed a novel Ad vector, named Ad-E4-122aT, exhibiting higher and longer-term transgene expression and lower hepatotoxicity than conventional Ad vectors. We also elucidated the mechanisms underlying Ad vector-induced hepatotoxicity during the early phase using Ad-E4-122aT. Next, we examined the therapeutic effects of the genes of interest, namely zinc finger AN1-type domain 3 (ZFAND3), lipoprotein lipase (LPL), and lysophospholipid acyltransferase 10 (LPLAT10), on lifestyle-related diseases using Ad-E4-122aT. We showed that the overexpression of ZFAND3 in the liver improved glucose tolerance and insulin resistance. Liver-specific LPL overexpression suppressed hepatic lipid accumulation and improved glucose metabolism. LPLAT10 overexpression in the liver suppressed postprandial hyperglycemia by increasing glucose-stimulated insulin secretion. Furthermore, we also focused on foods to advance research on the pathophysiology and treatment of lifestyle-related diseases. Cranberry and calamondin, which are promising functional foods, attenuated the progression of MASLD/NAFLD. Our findings will aid the development of new therapeutic methods, including gene therapy, for lifestyle-related diseases such as T2DM and MASLD/NAFLD.

The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

An adeno-associated virus variant enabling efficient ocular-directed gene delivery across species.

Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In non-human primates (NHPs), rAAVs are administered via suprachoroidal injection at a higher dose. However, high doses of rAAVs tend to increase additional safety risks. Here, we present a novel AAV capsid (AAVv128), which exhibits significantly enhanced transduction efficiency for photoreceptors and retinal pigment epithelial (RPE) cells, along with a broader distribution across the layers of retinal tissues in different animal models (mice, rabbits, and NHPs) following intraocular injection. Notably, the suprachoroidal delivery of AAVv128-anti-VEGF vector completely suppresses the Grade IV lesions in a laser-induced choroidal neovascularization (CNV) NHP model for neovascular age-related macular degeneration (nAMD). Furthermore, cryo-EM analysis at 2.1 Å resolution reveals that the critical residues of AAVv128 exhibit a more robust advantage in AAV binding, the nuclear uptake and endosome escaping. Collectively, our findings highlight the potential of AAVv128 as a next generation ocular gene therapy vector, particularly using the suprachoroidal delivery route.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

[In vitro and in vivo validation of cytotoxicity of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells].

Objective To evaluate the toxicology of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells and to provide a safety basis for the clinical evaluation of HER2-CAR-T cell therapy. Methods The recombinant lentiviral vector was used to generate HER2-CAR-T cells. Soft agar colony formation assay was used to observe the colony formation of HER2-CAR-T cells, and the colony formation rate was statistically analyzed. The HER2-CAR-T cell suspension was co-incubated with rabbit red blood cell suspension, and the hemolysis of red blood cells was evaluated by direct observation and microplate reader detection. The HER2-CAR-T cell preparation was injected into the ear vein of male New Zealand rabbits, and the stimulating effect of HER2-CAR-T cells on the blood vessels of the animals was observed by staining of tissue sections. The vesicular stomatitis virus envelope glycoprotein (VSV-G) gene of pMD 2.G vector was used as the target sequence, and the safety of the lentiviral vector was verified by real-time fluorescence quantitative PCR. The heart, liver, lung, and kidney of mice receiving HER2-CAR-T cell infusion were collected, and the lesions were observed by HE staining. Results The HER2-CAR-T cells were successfully prepared. These cells did not exhibit soft agar colony formation ability in vitro, and the HER2-CAR-T cell preparation did not cause hemolysis in New Zealand rabbit red blood cells. After the infusion of HER2-CAR-T cells into the ear vein of New Zealand rabbits, no obvious vascular stimulation response was found, and no specific amplification of VSV-G was detected. No obvious lesions were found in the heart, liver, lung and kidney tissues of the treatment group. Conclusion The prepared HER2-CAR-T cells have reliable safety.

Comparative in vivo characterization of newly discovered myotropic adeno-associated vectors.

BACKGROUND: Adeno-associated virus (AAV)-based gene therapy is a promising strategy to treat muscle diseases. However, this strategy is currently confronted with challenges, including a lack of transduction efficiency across the entire muscular system and toxicity resulting from off-target tissue effects. Recently, novel myotropic AAVs named MyoAAVs and AAVMYOs have been discovered using a directed evolution approach, all separately demonstrating enhanced muscle transduction efficiency and liver de-targeting effects. However, these newly discovered AAV variants have not yet been compared. METHODS: In this study, we performed a comparative analysis of these various AAV9-derived vectors under the same experimental conditions following different injection time points in two distinct mouse strains. RESULTS: We highlight differences in transduction efficiency between AAV9, AAVMYO, MyoAAV2A and MyoAAV4A that depend on age at injection, doses and mouse genetic background. In addition, specific AAV serotypes appeared more potent to transduce skeletal muscles including diaphragm and/or to de-target heart or liver. CONCLUSIONS: Our study provides guidance for researchers aiming to establish proof-of-concept approaches for preventive or curative perspectives in mouse models, to ultimately lead to future clinical trials for muscle disorders.

Engineered nanoparticles promote cardiac tropism of AAV vectors.

Cardiac muscle targeting is a notoriously difficult task. Although various nanoparticle (NP) and adeno-associated viral (AAV) strategies with heart tissue tropism have been developed, their performance remains suboptimal. Significant off-target accumulation of i.v.-delivered pharmacotherapies has thwarted development of disease-modifying cardiac treatments, such as gene transfer and gene editing, that may address both rare and highly prevalent cardiomyopathies and their complications. Here, we present an intriguing discovery: cargo-less, safe poly (lactic-co-glycolic acid) particles that drastically improve heart delivery of AAVs and NPs. Our lead formulation is referred to as ePL (enhancer polymer). We show that ePL increases selectivity of AAVs and virus-like NPs (VLNPs) to the heart and de-targets them from the liver. Serotypes known to have high (AAVrh.74) and low (AAV1) heart tissue tropisms were tested with and without ePL. We demonstrate up to an order of magnitude increase in heart-to-liver accumulation ratios in ePL-injected mice. We also show that ePL exhibits AAV/NP-independent mechanisms of action, increasing glucose uptake in the heart, increasing cardiac protein glycosylation, reducing AAV neutralizing antibodies, and delaying blood clearance of AAV/NPs. Current approaches utilizing AAVs or NPs are fraught with challenges related to the low transduction of cardiomyocytes and life-threatening immune responses; our study introduces an exciting possibility to direct these modalities to the heart at reduced i.v. doses and, thus, has an unprecedented impact on drug delivery and gene therapy. Based on our current data, the ePL system is potentially compatible with any therapeutic modality, opening a possibility of cardiac targeting with numerous pharmacological approaches.

Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo.

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

A universal recombinant adenovirus type 5 vector-based COVID-19 vaccine.

A universal recombinant adenovirus type-5 (Ad5) vaccine against COVID19 (Ad-US) was constructed, and immunogenicity and broad-spectrum of Ad5-US were evaluated with both intranasal and intramuscular immunization routes. The humoral immune response of Ad5-US in serum and bronchoalveolar lavage fluid were evaluated by the enzyme-linked immunosorbent assay (ELISA), recombinant vesicular stomatitis virus based pseudovirus neutralization assay, and angiotensin-converting enzyme-2 (ACE2) -binding inhibition assay. The cellular immune response and Th1/Th2 biased immune response of Ad5-US were evaluated by the IFN-γ ELISpot assay, intracellular cytokine staining, and Meso Scale Discovery (MSD) profiling of Th1/Th2 cytokines. Intramuscular priming followed by an intranasal booster with Ad5-US elicited the broad-spectrum and high levels of IgG, IgA, pseudovirus neutralizing antibody (PNAb), and Th1-skewing of the T-cell response. Overall, the adenovirus type-5 vectored universal SARS-CoV-2 vaccine Ad5-US was successfully constructed, and Ad5-US was highly immunogenic and broad spectrum. Intramuscular priming followed by an intranasal booster with Ad5-US induced the high and broad spectrum systemic immune responses and local mucosal immune responses.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.