Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease

Abstract

Gene therapy with genetically modified human CD34+ hematopoietic stem cells (HSCs) may be safer using targeted integration (TI) of transgenes into a genomic ‘safe harbor’ site than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno associated virus (AAV) 6 delivery of donor constructs in human HSCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus-positive HSCs with 6–16% human cell marking were observed following engraftment into mice. In HSCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 in resulted in ~15% gp91phox expression and increased NADPH oxidase activity in ex vivo–derived neutrophils. In mice transplanted with corrected HSCs, 4–11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction

“…Our findings from these targeting studies demonstrated a pronounced intronic requirement for CYBB cDNA expression from the endogenous CYBB promoter that was not evident in CYBB expression constructs utilizing other promoters, as we detected no gp91 phox protein expression from the endogenous promoter unless intron 1 was retained in the transcription unit, whereas the same cDNA was shown to be efficiently expressed in HSPCs from either an intron-less EF1a short promoter in a lentiviral expression vector in the current study or from an intron-less synthetic MND promoter after targeted genomic insertion into the AAVS1 safe-harbor site in a previous study. 26 It is not clear whether the CYBB promoter merely requires intronic splicing for effective expression or whether there are necessary transcriptional enhancer elements in CYBB intron 1, although a recent publication by Frazão et al 27 suggests a possible NF-kB enhancer element in intron 1, based on the association of a distant upstream NF-kB enhancer site with regions of the CYBB promoter and CYBB introns 1 and 2, and the finding that CYBB mRNA expression was reduced upon inhibition of NF-kB activity. Regardless of the particular intron-associated mechanism involved in CYBB expression, these observations highlight an important general design issue for effective gene targeting and knockin strategies-that donor constructs or targeting strategies may require inclusion or retention of intronic elements in order to achieve therapeutic or even detectable levels of transgene expression from an endogenous promoter.…”

Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease

“…This is inline with recent reports that have described efficient CD34 1 HPC targeted corrections or insertions. [19][20][21][22] We demonstrated our gene-targeting correction in iPSCs from 3 subjects and in HPCs from a fourth subject with p47-CGD. Three of these patients had the common homozygous DGT mutation at the NCF1 locus while one patient (subject 5; the source of iPSC of the P47-05 correction series) had normal GTGT at the start of exon 2 of NCF1 and instead had a homozygous D734-748 mutation in exon 8 of NCF1.…”

A New Chapter on Targeted Gene Insertion for X-CGD: Do Not Skip the Intro(n)

“…Several viral vectors, including adenovectors and adeno-associated vectors (AAVs), have been used to shuttle genes into cells of the inner ear, 1,2 but relatively low transfection efficiency continues to be an obstacle. 3,4 In this issue of Molecular Therapy, György et al 5 now show that AAV1 vectors linked to exosomes (exo-AAV1) achieve highly efficient transfection of all types of sensory cells (hair cells) within both the auditory and vestibular sensory epithelia of the inner ear.…”

“…This approach has been used, for example, to insert minigenes and, among those, the CYBB minigene into the AAVS1 locus, a putative genomic safe harbor that is also resistant to silencing. 2,3,5 Although, in principle, safe, this strategy is still not perfect, for there is only a partial rescue of NAPDH-oxidase function in fully differentiated neutrophils. Alternatively, to capture the advantages of natural gene regulatory mechanisms, scientists can use engineered nucleases to insert a functional minigene near the transcription start site of the disease-causing gene, also taking advantage of the increased cutting efficiency of the nuclease in this part of the gene structure.…”

“…Like many monogenic disorders of the immune system, X-CGD is amenable to gene therapy through gene addition. More recently, scientists have also started to explore nuclease-mediated targeted gene insertion as a strategy to restore gp91phox expression in reprogrammed induced pluripotent stem cells (iPSCs) [2][3][4] and hematopoietic stem and progenitor cells (HSPCs) 5 from CGD patients. In this issue of Molecular Therapy, Sweeny et al, 6 show that nucleasemediated insertion of a CYBB minigene into exon 2, but surprisingly not exon 1, can restore gp91phox protein expression in iPSC-derived granulocytes from X-CGD patients ( Figure 1).…”