Biology subjects

Afzal, S.

Publications and source records attributed to Afzal, S..

4 recordsLinked to original sources

bioRxiv2026

CRISPR-dPCR: A unified multiplex digital PCR framework for quantitative characterization of targeted knock-in

Accurate characterization of CRISPR knock-in requires distinguishing site-specific integration from total donor abundance and donor-derived background while measuring editing at the target site. Here, we present CRISPR-dPCR, a modular multiplex digital PCR framework combining a locked nucleic acid-enhanced cut-site assay with junction-based detection and two orthogonal short-amplicon strategies: Poisson linkage and homology-arm subtraction. We evaluated assay modules across genomic loci, neuroblastoma models and primary human T cells, with selected assays compared on two dPCR platforms. In genotype-defined clones and mixtures with low residual donor, homology-arm subtraction most closely recovered expected knock-in abundance. Linkage provided highly specific evidence of target-cargo association without junction-spanning amplification, whereas junction assays supplied boundary-specific confirmation but were more sensitive to amplicon length and multiplexing. Five-channel panels integrated cut-site editing, donor abundance, copy-number normalization and site-specific knock-in in a single reaction. Residual donor DNA limited quantitative interpretation, particularly in early post-editing samples. Modular extensions quantified defined unintended outcomes, including chromosomal translocations. CRISPR-dPCR integrates complementary measurements into a scalable analytical framework for genome-editing research, cell and gene therapy product development and applications in longitudinal clinical monitoring of targeted integration and transgene persistence.

molecular biology↗

bioRxiv2026

A Non-Viral CRISPR/Cas9 HDR Platform for Stable Engineering of Solid Tumor Models.

Virus-free genome engineering provides a flexible alternative to viral vectors for generating genetically modified cell models. Here, we establish an integrated biosafety level 1-compatible CRISPR/Cas9 homology-directed repair (HDR) workflow for stable transgene knock-in in neuroblastoma cell lines using non-viral delivery approaches. We systematically evaluated donor cassette architecture and delivery conditions across electroporation-based Cas9 ribonucleoprotein (RNP) delivery and lipid nanoparticle (LNP)-mediated co-delivery of Cas9 mRNA, sgRNA, and donor DNA. Modular AAVS1-targeting donor constructs identified a compact EF1(s)-Donor-Q8-Tag-sPA cassette that consistently yielded the strongest HDR-associated knock-in readouts, achieving up to 60% stable reporter-positive cells following electroporation without HDR enhancers. While LNP-mediated delivery enabled efficient CRISPR cargo co-delivery and generation of genetically modified tumor cell populations, knock-in efficiencies remained lower than those observed with electroporation. Subsequent enrichment approaches enabled generation of highly pure edited cell populations following both delivery strategies. Functional validation demonstrated stable transgene expression in vitro, including in three-dimensional bioprinted tumor models, and in vivo in xenograft mice without impairing tumor growth or viability. Together, these findings establish a practical non-viral HDR platform for stable engineering of solid tumor models and provide a framework for further optimization of genome editing workflows across distinct delivery modalities. Key findings- We establish a complete, virus-free CRISPR/Cas9 HDR workflow that reliably enables stable knock-in in solid tumor cell lines, demonstrated here in two neuroblastoma models under biosafety level 1 conditions. - We establish and evaluate LNP-mediated co-delivery of Cas9 mRNA, gRNA, and donor DNA for non-viral HDR knock-in in solid tumor models, revealing delivery modality-specific differences in editing efficiency, toxicity, and expression dynamics. - By systematically varying donor architectures, we identify a compact HDR template - combining a shortened custom EF1 promoter, the minimal Q8 surface reporter, and a synthetic polyadenylation signal (sPA) - that markedly improves knock-in efficiency in solid tumor cell lines, outperforming conventional cassettes. - Virus-free edited tumor cells generated using this workflow retain stable transgene expression and functional fitness in 3D bioprinted tumor constructs and xenograft mouse models, directly linking in vitro knock-in optimization to in vivo relevance. - The resulting biosafety level 1 compatible, end-to-end pipeline - integrating donor design, digital PCR-based quantification of precise integration, and enrichment strategies-offers a practical and transferable platform for engineering transgenic solid tumor models without viral vectors.

bioengineering↗

bioRxiv2025

Personalized CRISPR Knock-In Cytokine Gene Therapy to Remodel the Tumor Microenvironment and Enhance CAR T Cell Therapy in Solid Tumors

The immunosuppressive tumor microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumors. To address this, we developed a novel gene therapeutic strategy that enables localized remodeling of the TME via tumor-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumor-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma--a pediatric solid tumor with a suppressive TME--as a model, we applied CancerPAM to sequencing data from cell lines and patients to identify optimal integration sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG). CRISPR-mediated CXCL10 knock-in into tumor cells significantly enhanced CAR T cell infiltration and antitumor efficacy both in vitro and in vivo. In vivo, CXCL10-expressing tumors showed significantly increased early CAR T cell infiltration and prolonged survival compared to controls. CancerPAM rankings correlated strongly with target-site specificity and knock-in efficiency, validating its predictive performance. Our findings establish CancerPAM as a powerful tool for safe and effective CRISPR-based interventions and provide a conceptual framework for integrating cytokine-driven TME remodeling with cellular immunotherapies. This personalized strategy holds promise for enhancing CAR T cells and other immunotherapies across immune-refractory solid tumors.

bioengineering↗

bioRxiv2020

Renoprotective and haemodynamic effects of adiponectin and peroxisome proliferator-activated receptor agonist, pioglitazone, in renal vasculature of diabetic Spontaneously hypertensive rats

Pioglitazone, a therapeutic drug for diabetes, possesses full PPAR-{gamma} agonist activity and increase circulating adiponectin plasma concentration. Plasma adiponectin concentration decreases in hypertensive patients with renal dysfunctions. Present study investigated the reno-protective, altered excretory functions and renal haemodynamic responses to adrenergic agonists and ANG II following separate and combined therapy with pioglitazone in diabetic model of hypertensive rats. Pioglitazone was given orally [10mg/kg/day] for 28 days and adiponectin intraperitoneally [2.5{micro}g/kg/day] for last 7 days. Groups of SHR received either pioglitazone or adiponectin in combination. A group of Wistar Kyoto rats [WKY] served as normotensive controls, whereas streptozotocin administered SHRs served as diabetic hypertensive rats. Metabolic data and plasma samples were taken on day 0, 8, 21 and 28. In acute studies, the renal vasoconstrictor actions of Angiotensin II [ANGII], noradrenaline [NA], phenylephrine [PE] and methoxamine [ME] were determined. Diabetic SHRs control had a higher basal mean arterial blood pressure than the WKY, lower RCBP and plasma adiponectin, higher creatinine clearance and urinary sodium excretion compared to WKY [all P<0.05] which were normalized by the individual drug treatments and to greater degree following combined treatment. Responses to intra-renal administration of NA, PE, ME and ANGII were larger in diabetic SHR than WKY and SHRs [P<0.05]. Adiponectin significantly blunted responses to NA, PE, ME and ANG II in diabetic treated SHRs by 40%, whereas the pioglitazone combined therapy with adiponectin further attenuated the responses to adrenergic agonists by 65%. [all P <0.05]. These findings suggest that adiponectin possesses renoprotective effects and improves renal haemodynamics through adiponectin receptors and PPAR-{gamma} in diabetic SHRs, suggesting that synergism exists between adiponectin and pioglitazone. A cross-talk relationship also supposed to exists between adiponectin receptors, PPAR-{gamma} and alpha adrenoceptors in renal vasculature of diabetic SHRs.

pharmacology and toxicology↗