Adenovirus vectors in hematopoietic stem cell genome editing

Abstract

Genome editing of hematopoietic stem cells (HSCs) represents a therapeutic option for a number of hematological genetic diseases, as HSCs have the potential for self-renewal and differentiation into all blood cell lineages. This review presents advances of genome editing in HSCs utilizing adenovirus vectors as delivery vehicles. We focus on capsid-modified, helper-dependent adenovirus vectors that are devoid of all viral genes and therefore exhibit an improved safety profile. We discuss HSC genome engineering for several inherited disorders and infectious diseases including hemoglobinopathies, Fanconi anemia, hemophilia, and HIV-1 infection by ex vivo and in vivo editing in transgenic mice, nonhuman primates, as well as in human CD34⁺ cells. Mechanisms of therapeutic gene transfer including episomal expression of designer nucleases and base editors, transposase-mediated random integration, and targeted homology-directed repair triggered integration into selected genomic safe harbor loci are also reviewed.

Keywords: CRISPR/Cas9; HSCs; adenovirus vector; base editors; genome editing; in vivo transduction; targeted integration.

Fig. 1.

Examples for genome engineering strategies using HDAd vectors. HDAd vectors do not contain any viral coding genes. Stuffer DNA sequences are usually included to make up the total genome size of 28–32 kb. The episomal nature of HDAds can be exploited for transient expression of nucleases (e.g., CRISPR/Cas9) and BEs (e.g., CBEs). Simultaneous editing of two target sites can be achieved by incorporating two guide RNAs (gRNA). An example of a SIN CRISPR vector is shown to demonstrate a strategy for controlling CRISPR duration. For integrating vectors using SB100× transposase, two vectors are co-delivered. One expresses the transgene (e.g., human γ-globin driven by β-globin LCR/promoter) flanked by two FRT-IRs. The other vector expresses SB100× transposase and flippase. For HDR-mediated targeted integration, one donor template is flanked by two homology regions neighboring targeted genomic sites. The second vector is used to express CRISPR for creating DSBs. Ad/AAV hybrid vectors mediate targeted integration into AAVS1 locus. Two vectors are used to express transgene flanked by AAV ITRs and AAV Rep78, respectively. Episomal expression can be combined with integrated expression. As an example, cloning of CRISPR outside of the two FRT-IRs leads to transient expression of the nuclease system while concomitantly allowing the γ-globin cassettes bracketed by two FRT-IRs for SB100×-mediated integration. 3′UTR, 3′ end UTR; APOBEC1, APOBEC1 cytidine deaminase; Cas9n, Cas9 nickase; EF1α, human elongation factor-1 α promoter; GOI, gene of interest; LCR, β-globin LCR; LHA, left HA; pA, polyA signal; RHA, right HA; U6, U6 promoter; UGI, uracil-DNA glycosylase inhibitor; ψ, packaging signal sequence.

Fig. 2.

Ex vivo VS in vivo HSC transduction. Ex vivo HSC gene transfer is started with harvesting white MNCs in blood by leukapheresis after mobilization. Alternatively, bone marrow cells can be collected by aspiration. HSPCs are usually enriched from total MNCs by selection for CD34⁺ cells. The HSPCs are then be transduced using viral vectors (usually SIN-LVs) carrying transgenes. Modified cells are cryopreserved for shipment or if multiple harvesting/transductions are needed for accumulating more cells. Subsequently, the modified HSPCs are thawed, recovered, and infused back to the same patient following myeloablative conditioning. For in vivo HSC transduction, viral vectors (e.g., HDAd5/35++ vectors) expressing transgenes are administered intravenously following mobilization of HSCs. HSCs egressed from bone marrow are transduced in blood. Shortly following transduction, the modified cells home back to bone marrow and persist long-term.