In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods

Abstract

Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34 + mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro , rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12–14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ −/− Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSC s . Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.

Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality

“…Endogenous gene network repurposing becomes now possible with the recent advances of gene editing technics 66 and DNA template vectorization methods (single and double DNA templates 31,34,[67][68][69] , and AAV6-embeded template 26,29,32,70 ). Our work illustrates the therapeutic potential of this approach by rewiring different elements of the TCR activation pathway, but is not limited to it.…”

Neutralizing Antibodies Protect against Oral Transmission of Lymphocryptovirus

“…Eight to nine weeks post-challenge, mice were euthanized and single cell suspensions of splenocytes were collected and immunophenotyping of B cells using hCD45-FITC, mCD45-APC, CD19-PE-Cy7, hCD38-PerCP-Cy5.5, and hCD24-BV605, or T cells using hCD45-FITC at a 1:100 dilution, mCD45-APC at a 1:500 dilution, hCD4-Alexafluor647 at a 1:100 dilution, hCD8-PerCP-Cy5.5 at a 1:100 dilution, hCD137-BV421 at a 1:50 dilution and hCD69-BV605 at a 1:50 dilution, was performed by flow cytometry as described previously. 69 DNA was extracted from 5 million total splenocytes, utilizing the DNeasy Blood & Tissue Kit (QIAGEN) and according to the manufacturer’s instructions, for subsequent viral load analysis.…”

Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells

“…Cas9/AAV6-mediated genome editing is a robust system capable of introducing large genomic integrations at high frequencies across many loci in a wide variety of cell types, including HSPCs [18][19][20][21][22][23] . In fact, CRISPR-mediated approaches have been successfully employed to correct the disease-causing SNPs at high frequencies in HSPCs [22][23][24][25][26][27] . However, β-thalassemia is caused by lossof-function mutations scattered throughout the gene, rather than the single polymorphism responsible for SCD.…”