@article {6593, title = {SMART-lipid nanoparticles enabled mRNA vaccine elicits cross-reactive humoral responses against the omicron sub-variants.}, journal = {Mol Ther}, year = {2024}, month = {2024 Feb 27}, abstract = {

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has necessitated the development of broad cross-reactive vaccines. Recent findings suggest that enhanced antigen presentation could lead to cross-reactive humoral responses against the emerging variants. Toward enhancing the antigen presentation to dendritic cells (DCs), we developed a novel shikimoylated mannose receptor targeting lipid nanoparticle (SMART-LNP) system that could effectively deliver mRNAs into DCs. To improve the translation of mRNA, we developed spike domain-based trimeric S1 (TS1) mRNA with optimized codon sequence, base modification, and engineered 5{\textquoteright} and 3{\textquoteright} UTRs. In a mouse model, SMART-LNP-TS1 vaccine could elicit robust broad cross-reactive IgGs against Omicron sub-variants, and induced interferon-γ-producing T\ cells against SARS-CoV-2 virus compared with non-targeted LNP-TS1 vaccine. Further, T\ cells analysis revealed that SMART-LNP-TS1 vaccine induced long-lived memory T\ cell subsets, T helper 1 (Th1)-dominant and cytotoxic T\ cells immune responses against the SARS-CoV-2 virus. Importantly, SMART-LNP-TS1 vaccine produced strong Th1-predominant humoral and cellular immune responses. Overall, SMART-LNPs can be explored for precise antigenic mRNA delivery and robust immune responses. This platform technology can be explored further as a next-generation delivery system for mRNA-based immune therapies.

}, issn = {1525-0024}, doi = {10.1016/j.ymthe.2024.02.028}, author = {Mahalingam, Gokulnath and Rachamalla, Hari Krishnareddy and Arjunan, Porkizhi and Karuppusamy, Karthik V and Periyasami, Yogapriya and Mohan, Aruna and Subramaniyam, Kanimozhi and M, Salma and Rajendran, Vigneshwar and Moorthy, Mahesh and Varghese, George M and Mohankumar, Kumarasamypet M and Thangavel, Saravanabhavan and Srivastava, Alok and Marepally, Srujan} } @article {3340, title = {CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications.}, journal = {J Vis Exp}, year = {2022}, month = {2022 08 09}, abstract = {

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

}, keywords = {Animals, CRISPR-Cas Systems, Gene Editing, Genetic Therapy, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Mice}, issn = {1940-087X}, doi = {10.3791/64064}, author = {Venkatesan, Vigneshwaran and Christopher, Abisha Crystal and Karuppusamy, Karthik V and Babu, Prathibha and Alagiri, Manoj Kumar K and Thangavel, Saravanabhavan} } @article {2884, title = {Preferential Expansion of Human CD34CD133CD90 Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy.}, journal = {Hum Gene Ther}, volume = {33}, year = {2022}, month = {2022 02}, pages = {188-201}, abstract = {

CD34CD133CD90 hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34CD133CD90 HSCs over other subpopulations of adult HSPCs in culture. The preferential expansion enriches the HSCs in culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold . The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells . This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

}, keywords = {Animals, Antigens, CD34, Fetal Blood, Genetic Therapy, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Humans, Mice, Mice, Inbred NOD, Mice, SCID}, issn = {1557-7422}, doi = {10.1089/hum.2021.089}, author = {Christopher, Abisha Crystal and Venkatesan, Vigneshwaran and Karuppusamy, Karthik V and Srinivasan, Saranya and Babu, Prathibha and Azhagiri, Manoj Kumar K and Chambayil, Karthik and Bagchi, Abhirup and Rajendiran, Vignesh and Ravi, Nithin Sam and Kumar, Sanjay and Marepally, Srujan Kumar and Mohankumar, Kumarasamypet Murugesan and Srivastava, Alok and Velayudhan, Shaji R and Thangavel, Saravanabhavan} }