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Use of Serum-Free Medium with Fibronectin Fragment Enhanced Transduction in a System of Gas Permeable Plastic Containers to Achieve High Levels of Retrovirus Transduction at Clinical Scale

(summarized by Rachata Lumkul and Vivek Tanavde)

Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease which occurs in about five per million humans. The disease presents clinically with fungal and bacterial infections in multiple organs. In affected patients, neutrophils, monocytes, and eosinophils fail to destroy microbes utilizing SO and H₂O₂. Now that the relevant genes have been cloned, it is clear that there are four clinical/genetic subtypes of CGD. Each mutation affects a different chain of the phagocyte oxidase (phox) microbiocidal enzyme.

It is likely that even low efficiency gene therapy may have a therapeutic effect in CGD patients because granulocytes from some clinically normal female carriers of X-linked CGD have only 3%-5% of the normal phox enzymatic activity. With this rationale, Dr. Malech developed clinical scale methods for ex vivo transduction of mobilized peripheral blood CD34⁺ cells for gene therapy trials in CGD patients. In this symposium, he described the development and application of stem cell culture and retroviral transduction protocols using a closed system (gas-permeable plastic bloodbags), serum-free medium containing recombinant human hematopoietic growth factors, and fibronectin fragment-augmented retroviral transduction.

In Dr. Malech's phase I clinical trial, five patients with autosomal recessive p47^phox deficient CGD received intravenous infusion of autologous CD34⁺ peripheral blood stem cells (PBSC) which had been transduced ex vivo with a recombinant retrovirus encoding normal p47^phox (amphotropic MFGS-p47^phox retroviral vector). He used the Isolex 300 stem cell selection system (Nexell; Irvine, CA) to purify CD34⁺ cells from the leukapheresis cell product from G-CSF mobilized patients. Retroviral vector was harvested from producer lines cultured overnight in serum-free medium (BioWhittaker; Walkersville, MD; X-vivo 10 TM with 1% human serum albumin). The CD34⁺ cells were cultured and transduced in gas permeable flexible plastic containers (bloodbags) designed for stem cell culture (Nexell PL2417). The CD34⁺ cells were transduced twice, on days 2 and 3 of culture in serum-free medium containing recombinant human growth factor (interleukin 3 [IL-3]/GM-CSF fusion protein). Then the cells were washed and resuspended in plasmalyte containing 1% human serum albumin for intravenous administration to the patient. The patients did not receive any conditioning regimen.

Dr. Malech analyzed the transduction of patient blood cells and correction of phagocyte oxidase activity by using a phorbol 12-myristate 13-acetate (PMA)-stimulated nitroblue tetrazolium dye (NBT) test. A flow cytometry assay of oxidant production using dihydrorhodamine 123 (DHR) loading of the cells also was used to detect NADPH oxidase positive neutrophils in peripheral blood of patients after gene therapy.

The results of this first clinical trial detected phox⁺ granulocytes in peripheral blood of all five patients. Peak levels of corrected cells occured three to six weeks after infusion, and ranged from 0.004%-0.05% of total peripheral blood granulocytes. The %NBT⁺ colonies was between 9%-30%. Corrected cells were detectable for as long as six months after infusion, in some individuals. This trial also demonstrated the successful use of animal protein-free medium and a blood-bank-compatible closed system of gas permeable plastic containers for culture and transduction of PBSC.

In his second clinical trial, Dr. Malech studied gene therapy in the most common and severe form of CGD, X-linked gp91^phox deficiency. For each cycle of gene therapy, patients were given eight daily subcutaneous injections with the combination of 50 µg/kg flt-3 ligand and 5 µg/kg GM-CSF to mobilize CD34⁺ cells. Apheresis was performed when mobilization peaked at days 8-10. CD34⁺ cells were selected from leukapheresis products using the Isolex 300 (Nexell) which resulted in 80%-90% pure CD34⁺ cells at 60%-70% yield. Selected CD34⁺ cells were cultured and transduced in X-vivo TM 10 supplemented with 1% human serum albumin and growth factors (IL-3/GM-CSF fusion protein plus Flt-3 ligand). Dr. Malech prepared a high titer amphotropic MFGS-gp91^phox retrovirus vector. Culture and transduction were done in PL2417 containers coated with CH296 (Retronectin, Takara-Shuzo Corp.; Kyoto, Japan). CD34⁺ cells were exposed to serum -free retrovirus vector for seven h daily for four days, resulting in final transduction ranging from 48%-89% of colony-forming cells. By the end of this culture plus transduction, >70% of cells remained CD34⁺, and there was a >3-fold expansion of total cell numbers. Trial design and analysis were similar to the first trial. Three male patients with X-linked CGD each received two cycles of gene therapy without bone marrow conditioning; cycles were 50 days apart. In two patients, peak numbers of phox⁺ neutrophils were detected in peripheral blood (by the DHR flow cytometry assay) at three to four weeks after each cycle of gene therapy (1 in 500-1,700 cells; 0.2%-0.6%). In one patient receiving treatment for a fungal liver abscess, phox⁺ neutrophils were detected in pus draining from the abscess at four months after the first treatment. Two patients had >1 in 4,000 phox⁺ neutrophils in their peripheral blood. Thus, these improvements from the first trial resulted in a marked increase in ex vivo transduction efficiency. In one patient, peak levels of circulating phox⁺ neutrophils were >4-fold higher than the best results of the first trial.

In conclusion, Dr. Malech reports successful retroviral transduction in a clinical trial. Nevertheless, the current trials have achieved only low level, relatively short-term expression of the introduced gene. Further enhancements of the levels of stem cell transduction are anticipated in planned trials using additional hematopoietic growth factors (e.g., SCF, thrombopoietin, Flt-3 ligand) and/or alternative vectors (e.g., lentivirus).

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