Provenge is a novel immunotherapy agent that is safe and breaks tolerance to the tissue antigen PAP. Preliminary evidence for clinical efficacy warrants further exploration.

Gene repair of CD34 hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34 HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro. Transplant of gene-repaired X-CGD HSPCs into NOD (nonobese diabetic) SCID (severe combined immunodeficient) γc mice resulted in efficient engraftment and production of functional mature human myeloid and lymphoid cells for up to 5 months. Whole-exome sequencing detected no indels outside of the CYBB gene after gene correction. CRISPR-mediated gene editing of HSPCs may be applicable to other CGD mutations and other monogenic disorders of the hematopoietic system.

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.

Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N ¼ 5) or purified unstimulated NK cells from peripheral blood (N ¼ 4) resulted in good cell viability with eGFP expression in 85 ± 6% and 86 ± 4%, 24 h after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19 þ target cells resulting in X80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 h after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer. Cancer Gene Therapy ( IntroductionThe capacity of natural killer (NK) 1 cells to exert cytotoxicity against a variety of cancer cell types makes them an attractive tool for anti-cancer therapy. [2][3][4][5][6][7] Data gathered in the setting of allogeneic hematopoietic stem cell transplantation indicate that donor selection based on the degree of mismatch between expression of killer immunoglobulin-like receptors on donor NK cells and HLA Class I molecules expressed by the patient cells should maximize NK cell killing of target cells, 4,[8][9][10] hence augmenting the efficacy of hematopoietic stem cell transplantation. 6,7,11 In addition, it was reported that the infusion of haploidentical NK cells in a nonmyeloablative transplant setting could produce remissions in patients with acute myeloid leukemia. 5 Although NK cell cytotoxicity has a wide spectrum, some cancer cell types appear less susceptible or refractory to NK cell killing, because of failure to activate NK cells, induction of suppression or both. Among these relatively NKresistant cell types are lymphoid malignancies such as acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (B-CLL) and B-cell non-Hodgkin lymphoma. 3,[12][13][14][15][16] Chimeric antigen receptor has been studied since late 1980s. [17][18][19][20][21][22] It generally contains a single chain variable fragment as the extracellular antigen recognition unit and multiple lymphocyte activation domains as the intracellular activation part. Most work has been focused in arming T cells with this chimeric antigen receptor for antitumor effect 21-23 NK cells transduced with chimeric antigen receptor have also been exploited for anti-tumor effect. Various com...