Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models.

Sickle cell disease (SCD) is caused by a 20A > T mutation in the ß-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34+ cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact ß-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34+ cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus ß-to-ßs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34+ cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34+ HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.

MAGT1 messenger RNA-corrected autologous T and natural killer cells for potential cell therapy in X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection and neoplasia disease.

BACKGROUND AIM: X-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect' (XMEN) disease is caused by mutations in the magnesium transporter 1 (MAGT1) gene. Loss of MAGT1 function results in a glycosylation defect that abrogates expression of key immune proteins such as the NKG2D receptor on CD8+ T and NK cells, which is critical for the recognition and killing of virus-infected and transformed cells, a biomarker for MAGT1 function. Patients with XMEN disease frequently have increased susceptibility to EBV infections and EBV-associated B cell malignancies, for which no specific treatment options are currently available. Experimental transfer of donor EBV-specific cytotoxic T cells may be beneficial but carries the risks of eliciting alloimmune responses. An approach for cell therapy to address viral infections and associated complications that avoids the risks of alloimmunity is needed. METHODS: Here the authors assess the feasibility and efficiency of correcting autologous lymphocytes from XMEN patients by MAGT1 mRNA electroporation (EP) that avoids genomic integration and can be scaled for clinical application. RESULTS AND CONCLUSIONS: Restoration of NKG2D expression was demonstrated in XMEN patient lymphocytes after MAGT1 mRNA electroporation that reach healthy donor levels in CD8+ T and NK cells at 1-2 days after EP. NKG2D expression persisted at ∼50% for 2 weeks after EP. Functionally, mRNA-correction of XMEN NK cells rescued cytotoxic activity also to healthy donor NK cell level. The restored NKG2D receptor expression and function were unaffected by cryopreservation, which will make feasible repeat infusions of MAGT1 mRNA-corrected autologous XMEN CD8+ T and NK cells for potential short term therapy for XMEN patients without the risks of alloimmunization.

Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-Transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 109 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival. No significant off-target toxicities were observed. These data support further investigation of CARMA-hMeso as a potential treatment for ovarian cancer and other mesothelin-expressing cancers.

CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease.

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.

Large volume flow electroporation of mRNA: clinical scale process.

Genetic modification for enhancing cellular function has been continuously pursued for fighting diseases. Messenger RNA (mRNA) transfection is found to be a promising solution in modifying hematopoietic and immune cells for therapeutic purpose. We have developed a flow electroporation-based system for large volume electroporation of cells with various molecules, including mRNA. This allows robust and scalable mRNA transfection of primary cells of different origin. Here we describe transfection of chimeric antigen receptor (CAR) mRNA into NK cells to modulate the ability of NK cells to target tumor cells. High levels of CAR expression in NK cells can be maintained for 3-7 days post transfection. CD19-specific CAR mRNA transfected NK cells demonstrate targeted lysis of CD19-expressing tumor cells OP-1, primary B-CLL tumor cells, and autologous CD19+ B cells in in vitro assays with enhanced potency: >80% lysis at effector-target ratio of 1:1. This allows current good manufacturing practices (cGMP) and regulatory compliant manufacture of CAR mRNA transfected NK cells for clinical delivery.

Efficient large volume lentiviral vector production using flow electroporation.

Lentiviral vectors are beginning to emerge as a viable choice for human gene therapy. Here, we describe a method that combines the convenience of a suspension cell line with a scalable, nonchemically based, and GMP-compliant transfection technique known as flow electroporation (EP). Flow EP parameters for serum-free adapted HEK293FT cells were optimized to limit toxicity and maximize titers. Using a third generation, HIV-based, lentiviral vector system pseudotyped with the vesicular stomatitis glycoprotein envelope, both small- and large-volume transfections produced titers over 1×10(8) infectious units/mL. Therefore, an excellent option for implementing large-scale, clinical lentiviral productions is flow EP of suspension cell lines.

Delivery of whole tumor lysate into dendritic cells for cancer vaccination.

Results from multiple human studies have continued to spur the development of dendritic cells (DCs) as therapeutic vaccines for the treatment of cancer, chronic viral infections, and autoimmune diseases. The antigen-specific activity of DCs is dependent on the ability of the DCs to take up and process tumor-associated antigens for presentation to the immune system. Although immature DCs have been shown to naturally take up tumor-associated antigens by phagocytosis, approaches that significantly affect antigen delivery need further evaluation, especially if such methodologies can be demonstrated to result in the elicitation of more robust and comprehensive immune responses. We have developed a rapid, robust, scalable, and regulatory-compliant process for loading DCs with whole tumor lysate. The use of whole tumor lysate facilitates the generation of a more robust immune response targeting multiple unique antigenic determinants in patient's tumors and likely reduces the tumor's potential of immune escape. We demonstrate that DCs electroloaded with tumor lysate elicit significantly stronger antitumor responses both in a tumor challenge model and in a therapeutic vaccination model for preexisting metastasic disease. These effects are observed in a processing scheme that requires 20- to 40-fold lower amounts of tumor lysate when compared with the standard coincubation/coculture methods employed in loading DCs.

Efficient responses in a murine renal tumor model by electroloading dendritic cells with whole-tumor lysate.

Electroporation of dendritic cells (DCs) with tumor lysate elicited greater antitumor responses in vitro and in vivo, using less lysate than standard coincubation. Electroloaded DCs had normal surface marker expression and matured into competent antigen-presenting cells. In a renal carcinoma (RENCA) model, mice were pretreated with lysate-loaded DCs before tumor challenge. Mice that received DCs electroloaded with RENCA lysate had significantly smaller tumors (9+/-6 mm2) than mice given DCs coincubated with the same lysate (23+/-5 mm2). To evaluate a metastatic therapeutic tumor model, mice were first injected with Lewis lung carcinoma (LLC) and then given 2 doses of cryopreserved LLC lysate-loaded DCs. Mice treated with electroloaded DCs had a 50% reduction in lung metastases compared with control mice that received no DCs or DCs loaded with liver lysate. In contrast, DCs coincubated with LLC lysate were indistinguishable from controls. Tumor lysate-electroloaded but not-coincubated DCs also primed syngeneic mouse splenocytes in vitro to produce interferon-gamma and, specifically, lyse tumor cells. The electroloaded DCs elicited specific T-cell responses with less lysate than the amount reported in standard coincubation procedures. This approach may be particularly useful when small amounts of tumor material are available.

p53 and p14 increase sensitivity of gastric cells to H. pylori-induced apoptosis.

H. pylori infection of the gastric mucosa is associated with increased epithelial cell apoptosis. In vitro, interferon-gamma and TNF-alpha have been shown to increase the sensitivity of cells to apoptosis induced by H. pylori. The p53 tumor suppressor gene is frequently mutated in many cancers, including gastric cancer. Since p53 protein can induce apoptosis, we sought to determine whether or not p53 increases the ability of gastric epithelial cells to undergo apoptosis in response to H. pylori-induced cell injury. Human gastric epithelial cell lines, AGS (p53 wild-type) cells and AGS cells infected with HPV E6 gene (AGS-E6) to inactivate p53 were exposed to H. pylori. The p53, p21, and p14ARF proteins were measured in gastric epithelial cells by immunoelectrophoresis. Gastric epithelial cell apoptosis was measured by DNA end-labeling assay (TUNEL) and subG0 cell fractions using flow cytometry, and by agarose gel electrophoresis of DNA. Exposure to H. pylori increased the levels of p53, p21, and p14ARF proteins two fold in AGS cells. Gastric AGS cells with fragmented DNA increased from 1.1% to 68% in after exposure to H. pylori for 24 hr. However, AGS-E6 cells were relatively resistant to apoptosis induced by H. pylori (only 15% of cells underwent apoptosis). In additional experiments, mouse embryonic fibroblasts (MEFs) were used to further investigate the role of ARF in stabilizing p53 after exposure to H. pylori. Wild-type and p19ARF-/- MEFs were exposed to H. pylori and evaluated for activation of p53, p19ARF, and apoptosis. As with AGS cells, H. pylori stimulated a 2-fold increase in p53 and p19ARF in wild-type MEFs; however, there was no increase in p53 in ARF-null MEFs. H. pylori easily stimulated apoptosis in wild-type MEFs, although, the absence of p19ARF significantly reduced the ability of H. pylori to induce apoptosis in these cells. Activation of ARF by H. pylori is important in stabilizing p53 resulting in increased apoptosis. Thus, inactivation of either ARF or p53 in gastric cells may reduce their ability to undergo apoptosis in response to injury induced by H. pylori.

Highly efficient, large volume flow electroporation.

Electroporation is widely used to transfect and load cells with various molecules. Traditional electroporation using a static mode is typically restricted to volumes less than 1 mL, which limits its use in clinical and industrial bioprocessing applications. Here we report efficient, large volume transfection results by using a scalable-volume electroporation system. Suspended (Jurkat) and adherent cells (10T1/2 and Huh-7) were tested. A large macromolecule, FITC-conjugated dextran (MW=500 kD) was used to measure cell uptake, while a plasmid carrying the gene coding for enhanced green fluorescence protein (eGFP) was used to quantitate the flow electrotransfection efficiency as determined by flow cytometry. The flow electroloading efficiency of FITC-dextran was >90%, while the cell viability was highly maintained (>90%). High flow electrotransfection efficiency (up to 75%) and cell viability (up to 90%) were obtained with processing volumes ranging from 1.5 to 50 mL. No significant difference of electrotransfection efficiency was observed between flow and static electrotransfection. When 50 mL of cell volume was processed and samples collected at different time points during electroporation, the transgene expression and cell viability results were identical. We also demonstrated that DNA plasmid containing EBNA1-OriP elements from Epstein-Barr virus were more efficient in transgene expression than standard plasmid without the elements (at least 500 too 1000-fold increase in expression level). Finally, to examine the feasibility of utilizing flow electrotransfected cells as a gene delivery vehicle, 10T1/2 cells were transfected with a DNA plasmid containing the gene coding for mIL12. mIL12 transfected cells were injected subcutaneously into mice, and produced functional mIL12, as demonstrated by anti-angiogenic activity. This is the first demonstration of efficient, large volume, flow electroporation and the in vivo efficacy of flow electrotransfected cells. This technology may be useful for clinical gene therapy and large-scale bioprocesses.