Advances in Cell and Gene Therapy

Mendelian genetics contribute largely to the development of hypertension; therefore, the identification of genetic variants related to blood pressure (BP) regulation remains crucial and may reveal new therapeutic drug targets. The purpose of the present study was to screen the captive-bred Vervet colony for salt-sensitive sequence variants or single nucleotide polymorphisms (SNPs) in the selected Renin-Angiotensin-Aldosterone System (RAAS) genes associated with salt sensitivity. Blood samples were collected from 16 captive-bred Vervet monkeys for genotyping and gene expression analysis. The impact of the identified sequence variants was determined using online prediction tools. Sanger sequencing analysis revealed 21 sequence variants in AGT, CYP3A5, GRK4, and SCL4A5, of which 19 were novel and two were previously reported in humans. All novel variants were either predicted to be polymorphic, disease-causing, or possibly damaging by prediction tools. Furthermore, the mRNA expression for AGT was significantly higher in the normal BP group ( value = 0.02), and a similar trend was observed for CYP3A5 and GRK4, whereas SCL4A5 was higher in the hypertensive group. The identified salt-sensitive variants specifically in GRK4 may be suggestive to be the attributing factor of the elevated BP levels in these captive-bred Vervet monkeys. Therefore, RAAS variants could be considered as a biomarker to identify the potential risk of developing hypertension in both humans and nonhuman primates.

Immunotherapy for cancer treatment is growing at an unprecedented rate since the inception of chimeric antigen receptor T (CAR-T) cells. However, the efficacy of CAR-T cells against solid tumors is hampered by various issues, including “on-target, off-tumor toxicities,” T cell exhaustion, and immunosuppressive tumor microenvironment. To overcome these limitations, recent advances focus on optimizing CAR-T cells using vaccines to develop more effective cell immunotherapies. Here, we summarize the most recent studies on how vaccine-based CAR-T therapies are advancing the response of cancer immunotherapy as well as the current state of their clinical and preclinical development. Finally, we share perspectives on how future studies can incorporate other strategies to augment the antitumor response of vaccine-assisted CAR-T cell therapy.

In addition to laser photocoagulation, currently used therapeutic interventions for diabetic retinopathy (DR) include relatively short-lived anti-VEGF drugs targeting vascular endothelial growth factor (VEGF). The latter requires frequent administration via intravitreal injections to effect long-term VEGF suppression. However, due to the patient burden associated with this treatment modality, gene therapy may represent a preferable alternative, providing long-lasting yet patient-friendly effects. Here, we explore the therapeutic efficacy of rAAV2-sVEGFRv-1, a recombinant adeno-associated virus encoding a soluble variant of VEGF receptor-1, upon early DR processes. Bevacizumab, an anti-VEGF agent often prescribed off label to treat DR, was used as an experimental comparator. Administered by intravitreal injection to a streptozotocin-induced diabetic mouse model, rAAV2-sVEGFRv-1 was shown to effectively transduce the mouse retinas and express its transgene therein, leading to significant reductions in pericyte loss and retinal cell layer thinning, two processes that play major roles in DR progression. Acellular capillary formation, vascular permeability, and apoptotic activity, the latter being the cell death mechanism by which retinal neurodegeneration occurs, were also shown to be reduced by the therapeutic virus vector. Immunohistochemistry was used to visualize that rAAV2-sVEGFRv-1 has an effect on cell types important to DR pathophysiology, particularly the ganglion cell layer and glial cells. Combined with our previous work showing that the therapeutic virus vector reduces neovascularization, our current results reveal that rAAV2-sVEGFRv-1 addresses the early aspects of DR as well, thereby demonstrating its potential as a human gene therapeutic versus the condition as a whole.

One of the most important technologies in modern medicine is gene therapy, which allows therapeutic genes to be introduced into cells of the body. The approach involves genetics and recombinant DNA techniques that allow manipulating vectors for delivery of exogenous material to target cells. The efficacy and safety of the delivery system are a key step towards the success of gene therapy. Somatic cell gene therapy is the easiest in terms of technology and the least problematic in terms of ethics. Although genetic manipulation of germline cells at the gene level has the potential to permanently eradicate certain hereditary disorders, major ethical issues such as eugenics, enhancement, mosaicism, and the transmission of undesirable traits or side effects to patients’ descendants currently stymie its development, leaving only somatic gene therapy in the works. However, moral, social, and ethical arguments do not imply that germline gene therapy should be banned forever. This review discusses in detail the current challenges surrounding the practice of gene therapy, focusing on the moral arguments and scientific claims that affect the advancement of the technology. The review also suggests precautionary principles as a means to navigate ethical uncertainties.

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). Earlier studies reported on the copackaging of murine virus-like 30S RNA (VL30) genomes with -retroviral vectors generated in murine stable packaging cells. In an earlier study, VL30 mRNA was found to enhance the metastatic potential of human melanoma cells. These findings raise biosafety concerns regarding the possibility that therapeutic CAR-Ts have been inadvertently contaminated with potentially oncogenic VL30 retrotransposons. In this study, we demonstrated the presence of infectious VL30 particles in PG13 cell-conditioned media and observed the ability of these particles to deliver transcriptionally active VL30 genomes to human cells. Notably, VL30 genomes packaged by HIV-1-based vector particles transduced naïve human cells in culture. Furthermore, we detected the transfer and expression of VL30 genomes in clinical-grade CAR-T cells generated by transduction with PG13 cell-derived -retroviral vectors. Our findings raise biosafety concerns regarding the use of murine packaging cell lines in ongoing clinical applications.

Recombinant AAV (rAAV) gene therapy is being investigated as an effective therapy for several diseases including hemophilia B. Reports of liver tumor development in certain mouse models due to AAV treatment and genomic integration of the rAAV vector has raised concerns about the long-term safety and efficacy of this gene therapy. To investigate whether rAAV treatment causes cancer, we utilized two mouse models, inbred C57BL/6 and hemophilia B Balb/C mice (HemB), to test if injecting a high dose of various rAAV8 vectors containing or lacking hFIX transgene, a Poly-A sequence, or the CB or TTR promoter triggered liver fibrosis and/or cancer development over the course of the 6.5-month study. We observed no liver tumors in either mouse cohort regardless of rAAV treatment through ultrasound imaging, gross anatomical assessment at sacrifice, and histology. We did, however, detect differences in collagen deposition in C57BL/6 livers and HemB spleens of rAAV-injected mice. Pathology reports of the HemB mice revealed many pathological phenomena, including fibrosis and inflammation in the livers and spleens across different AAV-injected HemB mice. Mice from both cohorts injected with the TTR-hFIX vector demonstrated minimal adverse events. While not tumorigenic, high dose of rAAVs, especially those with incomplete genomes, can influence liver and spleen health negatively that could be problematic for cementing AAVs as a broad therapeutic option in the clinic.