Biology subjects

Basiri, M.

Publications and source records attributed to Basiri, M..

9 recordsLinked to original sources

bioRxiv2026

Implementation and Evaluation of Marker Rescue and qPCR Assays for Detection of Replication-Competent Retrovirus in Gene Therapy Products

Quality control of retroviral gene therapy vectors requires reliable detection of replication-competent retrovirus (RCR) particles. Cell-based assays remain the standard for detecting RCR, while PCR-based assays serve as rapid, complementary methods. Despite their widespread use, few studies provide comprehensive protocols and performance data to guide assay setup. Here we provide a detailed description and performance assessment of a cell-based firefly luciferase marker rescue assay for RCR testing. Additionally, we describe the setup and assessment of a qPCR-based RCR assay targeting retroviral pol gene sequence. Different viral copy numbers of MuLV were used to determine the final sensitivity of the marker rescue assay with and without initial amplification. Different q-PCR strategies were investigated for RCR testing using different types of primers, including env and pol genes. The marker rescue assay detected 73.2 RCR genome particles without amplification, while the amplification phase enabled detection of functional RCR particles and increased sensitivity, with the amplification step increasing viral particles by up to 5.23x103 fold. The PCR-based RCR assay using retroviral pol gene could detect approximately 7.32x103 viral particles. This study offers a resource for establishing RCR testing procedures for clinical-grade retroviral vector products.

cancer biology↗

bioRxiv2026

Assessment of Q-PCR based vector copy numbers and viral titration in CAR T cell products, Probe vs SYBR Green.

Assessment of vector copy numbers (VCNs) in comprised of the chimeric antigen receptor (CAR (T cells as a safety parameter and viral titration in the vector bank (VB) as an efficacy parameter are essential in the CAR T cell production procedure. Here, we developed a quantitative-PCR (Q-PCR) based VCN and viral titration analysis using both Probe and SYBR Green methods. Cellular viral titration was also applied for comparison of viral titration results. The VCN in CAR T cells with SYBR Green was 1.72, 2.31, and 2.46 respectively, while in the Probe based analysis, it was 1.33, 1.83, and 2.20. The molecular viral titration in SYBR Green resulted in 2.90*109, 5.24*1010, and 9.76*1010, while in the Probe it was 1.54*109, 1.13*109, and 7.73*108. The cellular titration resulted in 1.73*106, 2.16*106, and 2.49*106. In summary, both SYBR Green and Probe revealing that Probe strategy had a lower dependency on sample contamination and could be a better strategy for the Q-PCR method. The SYBR Green based viral titration method is significantly different from the Probe based method, with the Probe based method yielding better results compared to the cellular titration method. However, no significant difference was reported in both methods for VCNs.

immunology↗

bioRxiv2026

An ER Retrieval-and-Release System Reveals Ligand-Independent Basal Signaling by Cell Surface TCRs

The T cell receptor (TCR) machinery comprises the ligand-binding TCR{beta} and the CD3 signaling dimers. Individual subunits are synthesized in the endoplasmic reticulum (ER) and only the fully assembled TCRs reach the cell surface. We engineered a system to retained CD3{varepsilon} is in the ER. This allowed only partial TCR assembly, while continuous retrieval of CD3{varepsilon} from the Golgi back to the ER enabled complete assembly. Synchronous TCR release by biotin addition caused a transient increase in surface TCR abundance that gradually returned to steady-state levels. Mathematical modeling revealed that TCR levels were controlled by feedback mechanisms. Notably, surface-localized, but not ER-localized, TCRs spontaneously generated ligand-independent basal signaling. Phosphoproteomic analyses revealed that this signal is similar, but not identical, to ligand-induced TCR activation signals, with distinct phosphorylation patterns already evident at the CD3 subunits. Our findings establish the TCR retrieval-and-release system as a tool to investigate ligand-independent basal TCR signaling.

immunology↗

bioRxiv2024

Engineering CD3 Subunits with Endoplasmic Reticulum Retention Signal Facilitates Allogeneic CAR T Cell Production

The success of autologous CAR T cell therapies has driven interest in developing off-the-shelf allogeneic CAR T cells as a scalable and readily available option for broader patient access. Most of the current approaches involve the knockout of T cell receptor (TCR) subunits via genome editing for preventing graft-versus-host disease (GvHD). However, clinical translation of these methods faces challenges due to manufacturing complexities and emerging safety concerns like unintended long deletions and chromosomal loss. In this study, we explored an alternative approach by engineering synthetic CD3 subunits containing an endoplasmic reticulum retention (ERR) signal to suppress TCR surface expression by disrupting its trafficking to the plasma membrane. We screened multiple CD3-ERR candidate designs to identify the construct with the highest efficacy in TCR downregulation. The selected candidate, CD3{zeta}-ERR, was further characterized, demonstrating its ability to minimize TCR-mediated activation and alloreactivity without affecting T cell phenotype, cell cycle and cytokine-induced expansion. Subsequent assays revealed that CD3{zeta}-ERR CD19 CAR T cells retained their CAR-mediated cytotoxic function against CD19+ malignant cells. This study presents an alternative approach for TCR downregulation that circumvents genome editing. By using a transgene compatible with conventional viral vector delivery, this approach holds promise for scalable clinical-grade manufacturing of allogeneic CAR T cell therapies. Translational Impact StatementOur study introduces a scalable method to engineer allogeneic CAR T cells by reducing TCR expression without genome editing, thereby minimizing the risk of immune rejection (GvHD) while maintaining antitumor efficacy. This approach offers a practical and clinically translatable solution for producing "off-the-shelf" CAR T cell therapies, potentially broadening access to these life-saving treatments and streamlining their integration into existing clinical manufacturing processes.

immunology↗

bioRxiv2023

Characterization of CAR T Cells Manufactured using Genetically Engineered Artificial Antigen Presenting Cells

ObjectiveChimeric antigen receptor (CAR) T cell therapy has recently emerged as a promising approach for the treatment of different types of cancer. Improving CAR T cell manufacturing in terms of costs and product quality is an important concern for expanding the accessibility of this therapy. One proposed strategy for improving T cell expansion is to use genetically engineered artificial antigen presenting cells (aAPC) expressing a membrane-bound anti-CD3 for T cell activation. In this study, we characterized CAR T cells generated with this approach in terms of expansion efficiency, immunophenotype, and cytotoxicity. Materials and MethodsIn this experimental study, we generated an aAPC line by engineering K562 cells to express a membrane-bound anti-CD3 (mOKT3). T cell activation was performed by culturing PBMCs with either mitomycin C-treated aAPCs or surface-immobilized anti-CD3 and anti-CD28 antibodies. Untransduced and CD19-CAR-transduced T cells were characterized in terms of expansion, activation markers, IFN-{gamma} secretion, CD4/CD8 ratio, memory phenotype, and exhaustion markers. Cytotoxicity of CD19-CAR T cells generated by aAPCs and antibodies was also investigated using a bioluminescence-based co-culture assay. ResultsOur findings showed that the engineered aAPC line has the potential to expand CAR T cells similar to that of the antibody-based method. Although activation with aAPCs leads to a higher ratio of CD8+ and effector memory T cells in the final product, we did not observe a significant difference in IFN-{gamma} secretion cytotoxic activity or exhaustion between CAR T cells generated with aAPC or antibodies. ConclusionOur results show that despite the differences in the immunophenotypes of aAPC and antibody-based CAR T cells, both methods can be used to manufacture potent CAR T cells. These findings can be instrumental for the improvement of the T cell manufacturing process and future applications of aAPC-derived CAR T cells.

immunology↗

bioRxiv2023

CRISPR/Cas9-mediated Generation of COL7A1-deficient Keratinocyte Model of Recessive Dystrophic Epidermolysis Bullosa

ObjectiveRecessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blistering disease caused by mutations in the COL7A1 gene which is responsible for coding type VII collagen. Investigating the pathological mechanisms and novel candidate therapies for RDEB could be fostered by new cellular models. Here, we developed multiple immortalized COL7A1-deficient keratinocyte cell lines using CRISPR/Cas9 technology as RDEB cellular model. Materials and MethodsIn this experimental study, we used transient transfection to express COL7A1-targeting gRNA and Cas9 in HEK001 immortalized keratinocyte cell line followed by enrichment with fluorescent-activated cell sorting (FACS) via GFP expressing cells (GFP+ HEK001). Homogenous single-cell clones were then isolated, genotyped, and evaluated for type VII collagen expression. We performed a scratch assay to confirm the functional effect of COL7A1 knockout. ResultsWe achieved 46.1% (p < 0.001) efficiency of indel induction in the enriched transfected cell population. Except for 4% of single nucleotide insertions, the remaining indels were deletions of different sizes. Out of nine single clones expanded, two homozygous and two heterozygous COL7A1-deficient cell lines were obtained with defined mutation sequences. No off-target effect was detected in the knockout cell lines. Immunostaining and western blot analysis showed the lack of type VII collagen (COL7A1) protein expression in these cell lines. We also showed that COL7A1-deficient cells had higher motility compared with their wild-type counterparts. ConclusionWe reported the first isogenic immortalized COL7A1-deficient keratinocyte lines that provide a useful cell culture model to investigate aspects of RDEB biology and potential therapeutic options.

cell biology↗

bioRxiv2022

Generation and Functional Characterization of PLAP CAR-T Cells against Cervical Cancer Cells

Chimeric antigen receptor (CAR) T cell therapy is one of the cancer treatment modalities that has recently shown promising results in treating hematopoietic malignancies. However, one of the obstacles that need to be addressed in solid tumors is the on-target/off-tumor cytotoxicity due to the lack of specific tumor antigens with low expression in healthy cells. Placental alkaline phosphatase (PLAP) is a shared placenta/tumor-associated antigen (TAA) that is expressed in ovarian, cervical, colorectal, and prostate cancers and is negligible in normal cells. In this study, we constructed second-generation CAR T cells with a humanized scFv against PLAP antigen, then evaluated the characteristics of PLAP CAR T cells in terms of tonic signaling and differentiation in comparison with {Delta}PLAP CAR T cells and CD19 CAR T cells. In addition, by coculturing PLAP CAR T cells with HeLa cells, we analyzed the tumor-killing function and secretion of anti-tumor molecules. Results showed that PLAP CAR T cells not only could eliminate the cancer cells but also increase their proliferation in vitro. We also observed increased secretion of IL-2, granzyme A, and IFN-{gamma} by PLAP CAR T cells upon exposure to the target cells. In conclusion, PLAP CAR T cells are potential candidates for further investigation in cervical cancer and potentially other solid tumors.

immunology↗

bioRxiv2021

Pubertal sex hormones control transcriptional trajectories in the medial preoptic area

Pubertal maturation aids development of emotion, cognition, and reproduction. We investigated transcriptional dynamics in the medial preoptic area (MPOA), a hypothalamic center for reproductive behaviors, in male and female mice at single-cell resolution (scRNAseq) during puberty. Defined subsets of neurons expressing Slc32a1 and Esr1 (Vgat+ Esr1+) were the most transcriptionally dynamic compared to other cell types throughout puberty. These cell type specific transcriptional progressions towards adulthood were bidirectionally controlled by the levels of circulating testosterone and estradiol. Selective deletion of Esr1 in Slc32a1-expressing cells in the MPOA prior to puberty arrested transcriptional progression and revealed a sexually dimorphic gene-regulatory network governed by Esr1. Deletion of Esr1 in Vgat+ cells prevented the development of mating behavior in both sexes. These analyses reveal both sexually common and dimorphic transcriptional progressions during puberty as well as their regulatory mechanisms, which have important implications towards understanding adaptative and maladaptive processes governing adolescent brain development.

neuroscience↗

bioRxiv2020

Prepronociceptin expressing neurons in the extended amygdala encode and promote rapid arousal responses to motivationally salient stimuli

Motivational states are complex and consist of cognitive, emotional, and physiological components controlled by a network across multiple brain regions. An integral component of this neural circuitry is the bed nucleus of the stria terminalis (BNST). Here, we identified a subpopulation of neurons within BNST expressing the gene prepronociceptin (PnocBNST), that can modulate the rapid changes in physiological arousal that occur upon exposure to stimuli with motivational salience. Using in vivo two-photon calcium imaging we found that excitatory responses from individual PnocBNST neurons directly corresponded with rapid increases in pupillary size and occurred upon exposure to both aversive and rewarding odors. Furthermore, optogenetic activation of these neurons increased pupillary size, but did not alter approach/avoidance or locomotor behaviors. These findings suggest that excitatory responses in PnocBNST neurons encode rapid arousal responses irrespective of tested behaviors. Further histological, electrophysiological, and single-cell RNA sequencing data revealed that PnocBNST neurons are composed of genetically and anatomically identifiable subpopulations that can be further investigated. Taken together, our findings demonstrate a key role for a PnocBNST neuronal ensemble in encoding the rapid arousal responses that are triggered by motivational stimuli.

neuroscience↗