Analysis of hepatic lentiviral vector transduction; implications for preclinical studies and clinical gene therapy protocols.

Lentiviral vector-transduced T-cells were approved by the FDA as gene therapy anti-cancer medications. Little is known about the host genetic variation effects on the safety and efficacy of the lentiviral vector gene delivery system. To narrow this knowledge-gap, we characterized hepatic gene delivery by lentiviral vectors across the Collaborative Cross (CC) mouse genetic reference population.

The Updated Mouse Universal Genotyping Array Bioinformatic Pipeline Improves Genetic QC in Laboratory Mice.

The MiniMUGA genotyping array is a popular tool for genetic QC of laboratory mice and genotyping of samples from most types of experimental crosses involving laboratory strains, particularly for reduced complexity crosses. The content of the production version of the MiniMUGA array is fixed; however, there is the opportunity to improve array's performance and the associated report's usefulness by leveraging thousands of samples genotyped since the initial description of MiniMUGA in 2020. Here we report our efforts to update and improve marker annotation, increase the number and the reliability of the consensus genotypes for inbred strains and increase the number of constructs that can reliably be detected with MiniMUGA.

Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells.

For more than a decade, genetically engineered autologous T-cells have been successfully employed as immunotherapy drugs for patients with incurable blood cancers. The active components in some of these game-changing medicines are autologous T-cells that express viral vector-delivered chimeric antigen receptors (CARs), which specifically target proteins that are preferentially expressed on cancer cells. Some of these therapeutic CAR expressing T-cells (CAR-Ts) are engineered via transduction with -retroviral vectors (-RVVs) produced in a stable producer cell line that was derived from murine PG13 packaging cells (ATCC CRL-10686).

Human Tumor Targeted Cytotoxic Mast Cells for Cancer Immunotherapy.

The diversity of autologous cells being used and investigated for cancer therapy continues to increase. Mast cells (MCs) are tissue cells that contain a unique set of anti-cancer mediators and are found in and around tumors. We sought to exploit the anti-tumor mediators in MC granules to selectively target them to tumor cells using tumor specific immunoglobin E (IgE) and controllably trigger release of anti-tumor mediators upon tumor cell engagement.

Harnessing the Anti-Tumor Mediators in Mast Cells as a New Strategy for Adoptive Cell Transfer for Cancer.

The emergence of cancer immunotherapies utilizing adoptive cell transfer (ACT) continues to be one of the most promising strategies for cancer treatment. Mast cells (MCs) which occur throughout vascularized tissues, are most commonly associated with Type I hypersensitivity, bind immunoglobin E (IgE) with high affinity, produce anti-cancer mediators such as tumor necrosis factor alpha (TNF-α) and granulocyte macrophage colony-stimulating factor (GM-CSF), and generally populate the tumor microenvironments. Yet, the role of MCs in cancer pathologies remains controversial with evidence for both anti-tumor and pro-tumor effects.

Structural, functional, and immunogenicity implications of F9 gene recoding.

Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions.

A Single Synonymous Variant (c.354G>A [p.P118P]) in Confers Enhanced Specific Activity.

Synonymous variants within coding regions may influence protein expression and function. We have previously reported increased protein expression levels ex vivo (~120% in comparison to wild-type) from a synonymous polymorphism variant, c.354G>A [p.

Gene Delivery to Human Limbal Stem Cells Using Viral Vectors.

Limbal stem cell (LSC) transplantation is a promising treatment for ocular surface diseases especially LSC deficiency. Genetic engineering represents an attractive strategy to increase the potential for success in LSC transplantations either by correcting autologous diseased LSCs or by decreasing the immunogenicity of allogeneic LSCs. Therefore, two popular viral vectors, adeno-associated viral (AAV) vector and lentiviral (LV) vector, were compared for gene delivery in human LSCs.

Superior lentiviral vectors designed for BSL-0 environment abolish vector mobilization.

Lentiviral vector mobilization following HIV-1 infection of vector-transduced cells poses biosafety risks to vector-treated patients and their communities. The self-inactivating (SIN) vector design has reduced, however, not abolished mobilization of integrated vector genomes. Furthermore, an earlier study demonstrated the ability of the major product of reverse transcription, a circular SIN HIV-1 vector comprising a single- long terminal repeat (LTR) to support production of high vector titers.

Correction to: Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy.

The article "Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy", written by "Aaron B. Simmons, Colin A. Bretz, Haibo Wang, Eric Kunz, Kassem Hajj, Carson Kennedy, Zhihong Yang, Thipparat Suwanmanee, Tal Kafri and M.

Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy.

Inhibition of vascular endothelial growth factor (VEGF) in retinopathy of prematurity (ROP) raises concerns for premature infants because VEGF is essential for retinovascular development as well as neuronal and glial health. This study tested the hypothesis that endothelial cell-specific knockdown of VEGF receptor 2 (VEGFR2), or downstream STAT3, would inhibit VEGF-induced retinopathy without delaying physiologic retinal vascular development. We developed an endothelial cell-specific lentiviral vector that delivered shRNAs to VEGFR2 or STAT3 and a green fluorescent protein reporter under control of the VE-cadherin promoter.

Targeted Knockdown of Overexpressed VEGFA or VEGF164 in Müller cells maintains retinal function by triggering different signaling mechanisms.

Oxygen-induced retinopathy (OIR) upregulates Müller cell vascular endothelial growth factor A (VEGFA) that causes intravitreal neovascularization similar to severe retinopathy of prematurity (ROP). Safety concerns exist with anti-VEGF treatment for ROP. We evaluated long-term knockdown of Müller cell-VEGFA with short-hairpin RNAs to VEGFA or VEGF via subretinal lentivirus delivery (L-VEGFAshRNA, L-VEGF164shRNA) on retinal structure and function in a rat OIR model.

Toward Personalized Gene Therapy: Characterizing the Host Genetic Control of Lentiviral-Vector-Mediated Hepatic Gene Delivery.

The success of lentiviral vectors in curing fatal genetic and acquired diseases has opened a new era in human gene therapy. However, variability in the efficacy and safety of this therapeutic approach has been reported in human patients. Consequently, lentiviral-vector-based gene therapy is limited to incurable human diseases, with little understanding of the underlying causes of adverse effects and poor efficacy.

Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects.

Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)-induced central nervous system damage. However, all HSPCT-treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor-derived cells and 2) insufficient uptake of donor cell-secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation-independent mannose 6-phosphate-receptor (CI-MPR).

Insights into the Pathogenesis and Treatment of Krabbe Disease.

Krabbe disease (globoid cell leukodystrophy, GLD) is an inherited disease caused by a deficiency in the lysosomal enzyme galactocerebrosidase (GALC). The major galactosylated lipid degraded by GALC is galactosylceramide. However, GALC is also responsible for the degradation of galactosylsphingosine (psychosine), a highly cytotoxic glycolipid.

Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors.

The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models.

Functional analysis of the putative integrin recognition motif on adeno-associated virus 9.

Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown.

CCR5 Gene Editing of Resting CD4(+) T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice.

CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4(+) T cells.

Quantitative analyses of retinal vascular area and density after different methods to reduce VEGF in a rat model of retinopathy of prematurity.

Purpose: Targeted inhibition of Müller cell (MC)-produced VEGF or broad inhibition of VEGF with an intravitreal anti-VEGF antibody reduces intravitreal neovascularization in a rat model of retinopathy of prematurity (ROP). In this study, we compared the effects of these two approaches on retinal vascular development and capillary density in the inner and deep plexi in the rat ROP model.

Methods: In the rat model of ROP, pups received 1 μL of (1) subretinal lentivector-driven short hairpin RNA (shRNA) to knockdown MC-VEGFA (VEGFA.

Targeting Müller cell-derived VEGF164 to reduce intravitreal neovascularization in the rat model of retinopathy of prematurity.

Purpose: To determine whether knockdown of Müller cell-derived VEGFA-splice variant, VEGF164, which is upregulated in the rat retinopathy of prematurity (ROP) model, safely inhibits intravitreal neovascularization (IVNV).

Methods: Short hairpin RNAs for VEGF164 (VEGF164.shRNAs) or luciferase.

Evolutionary etiology of high-grade astrocytomas.

Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events.