Biology subjects

Adair, J. E.

Publications and source records attributed to Adair, J. E..

3 recordsLinked to original sources

bioRxiv2025

In vivo production of an anti-HIV antibody from primate hematopoietic cells by non-viral knock-in

Non-viral gene editing offers a practical alternative to viral delivery for durable biologics production. Clinical trials have shown that adeno-associated virus encoding broadly neutralizing antibodies can protect against HIV, but result in limited, short-lived responses. The development of non-viral gene editing approaches in hematopoietic stem and progenitor cells holds promise for long-term antibody production. In this study, we evaluated CRISPR/Cas9 and CRISPR/Cas12a for gene knock-in at the immunoglobulin heavy chain locus in non-human primate hematopoietic stem and progenitor cells. Delivering the nuclease as a protein alongside a custom DNA template, we optimized editing with Cas12a and demonstrated higher knock-in efficiency and fewer non-specific edits than Cas9. Transplantation of edited non-human primate hematopoietic stem and progenitor cells into MISTRG mice led to engraftment, B cell differentiation, and transgene expression of a reporter transgene or anti-HIV antibody after HIV immunization with detectable anti-HIV antibody titers in peripheral blood circulation. These findings demonstrate the feasibility of using non-viral gene editing in HSPC as a potential strategy for sustained biologics production in the treatment of chronic diseases such as HIV. Future work will assess the efficacy of this model in a non-human primate model of HIV infection.

immunology↗

bioRxiv2024

Cas9 RNP Physiochemical Analysis for Enhanced CRISPR-AuNP Assembly and Function

CRISPR therapy for hematological disease has proven effective for transplant dependent beta thalassemia and sickle cell anemia, with additional disease targets in sight. The success of these therapies relies on high rates of CRISPR-induced double strand DNA breaks in hematopoietic stem and progenitor cells (HSPC). To achieve these levels, CRISPR complexes are typically delivered by electroporation ex vivo which is toxic to HSPCs. HSPCs are then cultured in stimulating conditions that promote error-prone DNA repair, requiring conditioning with chemotherapy to facilitate engraftment after reinfusion. In vivo delivery by nanocarriers of CRISPR gene editing tools has the potential to mitigate this complexity and toxicity and make this revolutionary therapy globally available. To achieve in vivo delivery, the inherent restriction factors against oligonucleotide delivery into HSPCs, that make ex vivo manipulation including electroporation and stimulation essential, must be overcome. To this end, our group developed a CRISPR carrying gold nanoparticle (CRISPR-AuNP) capable of delivering either Cas9 or Cas12a CRISPRs as ribonucleoprotein complexes (RNP) without compromising HSPC fitness. However, the most commonly used CRISPR, Cas9, demonstrated inconsistent activity in this delivery system, with lower activity relative to Cas12a. Investigation of Cas9 RNP biophysics relative to Cas12a revealed duplex RNA instability during the initial loading onto Au cores, resulting in undetectable Cas9 loading to the particle surface. Here we demonstrate preformation of RNP before loading, coupled with optimization of the loading chemistry and conditions, resulted in 39.6 {+/-} 7.0 Cas9 RNP/AuNP without compromising RNP activity in both in vitro assays and primary human HSPC. The same alterations improved Cas12a RNP/AuNP loading 10-fold over previously reported levels. To achieve particle stability, the reported polyethyleneimine outer coating was altered to include PEGylation and the resulting 2nd generation CRISPR-AuNP demonstrates favorable nanoformulation characteristics for in vivo administration, with a hydrophilic, more neutral nanoparticle surface. Direct treatment of HSPC in vitro showed 72.5 {+/-} 7.37% uptake of 2nd generation CRISPR-AuNP in primary human HSPC, but with endosomal accumulation and low rates of gene editing consistent with low levels of endosomal escape.

bioengineering↗

bioRxiv2019

Sort-purification of human CD34+CD90+ cells reduces target cell population and improves lentiviral transduction

ABSTRACTHematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant and infectious diseases. We have shown in a nonhuman primate (NHP) HSC gene therapy and transplantation model that the CD34+CD90+ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. Here we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC-enrichment strategies include the purification of CD133+ cells or CD38low/- subsets of CD34+ cells from human blood products. We directly compared these strategies to the isolation of CD90+ cells using a GMP-grade flow-sorting protocol with clinical applicability. We show that CD90+ cell selection results in 40-fold fewer target cells in comparison to CD133+ or CD38low/- CD34 subsets without compromising the engraftment potential in vivo. Single cell RNA sequencing confirmed nearly complete depletion of lineage committed progenitor cells in CD90+ fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90+ cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and genome editing products.

cell biology↗