Narda Theobald

Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long-term correction of oxidase activity in peripheral blood neutrophils.

Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91(phox), achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was registered at www.clinicaltrials.gov as #NCT00394316.

Chromothriptic Cure of WHIM Syndrome

Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patients cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells.

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60(+)), with variable yield (6 to >500 TRA-1-60(+) iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP-reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34(+) HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14(+) monocytes do not yield iPSC colonies under these reprogramming conditions.

Human CD34 cell preparations contain over 100-fold greater NOD/SCID mouse engrafting capacity than do CD34 cell preparations

OBJECTIVE The CD34 cell surface marker is used widely for stem/progenitor cell isolation. Since several recent studies reported that CD34(-) cells also have in vivo engrafting capacity, we quantitatively compared the engraftment potential of CD34(+) vs CD34(-) cell preparations from normal human placental/umbilical cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PBSC) specimens, using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. METHODS CD34(+) and CD34(-) cell preparations were purified by four different approaches in 14 individual experiments involving 293 transplanted NOD/SCID mice. In most experiments, CD34(+) cells were depleted twice (CD34(=)) in order to obtain efficient depletion of CD34(+) cells from the CD34(-) cell preparations. RESULTS Dose-dependent levels of human hematopoietic cells were observed after transplantation of CD34(+) cell preparations. To rigorously assess the complementary CD34(-) cell preparations, cell doses 10- to 1000-fold higher than the minimum dose of the CD34(+) cell preparations necessary for engraftment were transplanted. Nevertheless, of 125 NOD/SCID mice transplanted with CD34(-) cell preparations purified from the same starting cells, only six mice had detectable human hematopoiesis, by flow cytometric or PCR assay. CONCLUSIONS CD34(-) cells provide only a minor contribution to hematopoietic engraftment in this in vivo model system, as compared to CD34(+) cells from the same samples of noncultured human cells. Hematopoiesis derived from actual CD34(-) cells is difficult to distinguish from that due to CD34(+) cells potentially contaminating the preparations.

41. Therapeutic Level CRISPR-Oligomer-Mediated Correction of X-CGD Patient Hematopoietic Stem Cells Using Non-Viral, cGMP Compliant, Scalable, and Closed System

Gene therapy using integrating viral vectors in hematopoietic stem cells (HSC) has shown clinical benefit in genetic diseases. However, there remain safety concerns associated with random integration and the lack of regulation of gene expression. Efficient and site-specific correction of mutation(s) in HSC using non-viral methods may improve safety and regulation of gene expression. Chronic granulomatous disease (CGD) due to defective phagocyte NADPH oxidase complex and lack of bactericidal superoxide and other reactive oxidative species (ROS) is characterized by severe infections and hyperinflammation. Although the X-linked form of CGD with gp91phox deficiency results from mutations that span the CYBB gene, we identified a ‘hotspot’ mutation at Exon 7 c. 676C>T, causing a premature stop codon in 17 out of 285 patients with X-linked CGD at the NIH. Here we report the result of the efficient correction of the hotspot CYBB mutation using highly efficient CRISPR (Cas9 and sgRNA) system with an oligomer as donor repair template, using MaxCytes commercially/clinically validated cGMP/regulatory compliant and closed platform technology. Plasmids encoding Cas9 and gRNA were purchased from the Genomic Engineering Center at Washington University (St. Louis, MO). The mRNA encoding Cas9 and gRNA were in vitro transcribed at MaxCyte using mMESSAGE mMACHINE® T7 Ultra kit, (Ambion, Austin, TX). We screened and selected best gRNA from four gRNA candidates for correction and then optimized transfection conditions with EBV-transformed B cell line (B-LCL) derived from an adult patient (P1) with the hotspot CYBB mutation. Transfected B-LCL exhibit 80±6% viability, minimal detectable toxicity as determined by cell proliferation rate referenced to control cells, and efficient site-specific gene correction with 20-50% WT gp91 expression. These developed protocols were used to treat G-CSF and pleraxifor mobilized CD34+ HSC from P1. Following optimization, in vitro treated HSC from P1 achieved 20-30% WT gp91 expression, with >50% viability, and minimal loss of cell proliferation capacity compared with control cells. CD34+ HSCs undergo myeloid differentiation in DMEM supplemented with G-CSF prior to functional evaluation using flow cytometric dihydrorhodamine (DHR) assay, demonstrating ~20% ROS+ cells in treated samples compared to ~80% in normal controls. P1s HSC treated the same way were transplanted into immunodeficient mice, and analyzed 8 weeks later. Bone marrow from mice transplanted with P1 treated cells showed CD45+ human cell engraftment rates at 50-80%, and of the forward/side scatter-gated granulocytes, 11-26% express gp91phox relative to 68% in normal control. Peripheral blood from mice demonstrated 11-23% human CD45+ cells, of which 9-21% expressed gp91phox, compared to 79% in normal controls. Deep sequencing of human CD45+ cells sorted from mouse bone marrow confirmed high rates (up to 21%) of genetic correction from the ‘T’ mutation to the wildtype ‘C’. Since female carriers of X-CGD with ~10-15% normal functioning neutrophils appear to have normal resistance to infections, this level of correction at 10-20% in human CD45+ cells from transplanted mice suggest CRISPR/oligo approach a feasible therapeutic option for treatment of CGD patients with the Ex7, c. 676C>T mutation.

54. Genome Editing of Primary Human CD34+ Hematopoietic Stem Cells Enables a Safe Harbor Targeted Gene Addition Therapeutic Strategy for Chronic Granulomatous Disease

Many monogenic recessive diseases of blood can, in principle, be cured by transfer of functional therapeutic transgene to the genome of the hematopoietic stem cell (HSC) – a strategy proven successful for multiple rare diseases using current integrating vector gene therapy. With a focus on X-linked chronic granulomatous disease (X-CGD), we report a directed approach orthogonal to randomly integrating retrovector gene therapy: the highly specific targeted placement of the curative transgene into a validated safe harbor locus in human HSCs via human genome editing with zinc finger nucleases (ZFNs) and donor insert delivery using an AAV6 vector.We describe here an integrated targeted delivery platform customized for targeted addition to human HSCs using a cGMP-compliant electroporation system compatible with clinical scale production. Using next-generation, highly optimized ZFNs against the AAVS1/PPP1R12C gene locus, we optimized conditions for addition of the fluorescent Venus cDNA into human peripheral blood G-CSF mobilized CD34+ HSCs. Venus expression in manipulated human HSCs in vitro reached >50% efficiency, while earlier experiments demonstrated persistence of gene-modified cells in NSG mice with 12-15% Venus+ human CD45+ cells retrieved from transplanted mouse bone marrow and overall human HSC engraftment levels of >15%. Targeted integration (TI) rates achieved in human CD45+ cells from mouse bone marrow were 28-57%. Similar levels of Venus+ (~10%) are observed in spleen and peripheral blood CD45+ cells, indicating differentiation of gene-modified CD34 HSCs into circulating blood cells.X-CGD patients suffer from severe bacterial and fungal infections with excessive inflammation due to a defect in the gp91phox subunit of phagocyte oxidase. To extend the results above to CGD, we therefore used the same approach to target addition of the relevant curative transgene, gp91phox, into the AAVS1 safe harbor locus of HSCs from patients with X-linked CGD. In vitro levels of gp91phox expression in gene-modified patient CD34+ HSCs population achieve 12-16% gp91phox expression by flow cytometric analysis, with an NSG xenograft study demonstrating 3-5% of the engrafted human CD45+ cells expressing gp91phox. Of note, the MND-driven gp91phox expression from the safe harbor locus in human neutrophils differentiating from CD34+ cells transplanted into the NSG mouse model parallels wildtype gp91phox levels produced at the native locus.Our studies demonstrate the feasibility of targeted addition of different genes at the AAVS1 safe harbor site of the genome in human HSCs at an unprecedented efficiency and specificity; we demonstrate the efficient correction of the enzymatic defect in neutrophils arising from patient-derived HSCs in vivo. In sum with the advances in GMP-scale cell processing for genome editing, and the charted regulatory path for ZFNs to the clinic provided by ongoing trials in HIV, our studies represent the foundation for a rapid translation of ZFN-driven targeted addition as a clinical modality for X-linked CGD.

298. Transfection of Neutrophils from X-Linked Chronic Granulomatous Disease Patients with gp91phox mRNA Restores Oxidase Activity with High Efficiency and Viability

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patients own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.