Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency.

Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34(+) cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc(scid)/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34(+) cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.

The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

Cytogenetics, immunostaining for fibroblast growth factors, p53 sequencing, and clinical features of two cases of cystosarcoma phyllodes.

BACKGROUND: We present cytogenetics and fibroblast growth factor immunohistochemistry in one case of cystosarcoma phyllodes with localized disease and one with metastatic spread. The p53 gene was sequenced in the malignant case. METHODS AND RESULTS: Karyotype analysis used trypsin-Giemsa banding. Immunohistochemistry of FGF1, FGF2, FGFR1 and p53 used avidin-biotin detection of the primary antibody. One case had a mosaic female karyotype and three clones: one normal, one with trisomy 7, and one with both trisomy 5 and a rearranged chromosome 1. In the second case, a resected pulmonary metastasis had the karyotype 43-47,XX,+mar1,+mar2[6]/43-46,XX, +del(7)(p10)[3],+mar2[1][cp3]/46,XX[10]. These tumors expressed FGF1, FGF2, and FGFR1. The malignant case showed immunostaining for p53 protein, but a wild-type gene sequence. CONCLUSION: The karyotype of cystosarcoma phyllodes is complex, with wide case-to-case variation. These tumors express members of the FGF family. Metastatic behavior can occur in the presence of a wild-type p53 gene.

Gene transfer approaches to the lysosomal storage disorders.

The work summarized in this paper used animal and cell culture models systems to develop gene therapy approaches for the lysosomal storage disorders. The results have provided the scientific basis for a clinical trial of gene transfer to hematopoietic stem cells (HSC) in Gaucher disease which is now in progress. The clinical experiment is providing evidence of HSC transduction, competitive engraftment of genetically corrected HSC, expression of the GC transgene, and the suggestion of a clinical response. In this paper we will review the progress made in Gaucher disease and include how gene transfer might be studied in other lysosomal storage disorders.

The effect of cationic liposome pretreatment and centrifugation on retrovirus-mediated gene transfer.

Pretreatment of retroviral supernatants with the cationic liposomes DOTMA-DOPE (Lipofectin), DC-Chol-DOPE and DOSPA-DOPE (Lipofectamine) was found to enhance static transductions of TF-1 target cells. The relative effectiveness at increasing transduction efficiencies (TE) was: DOSPA > DC-Chol > DOTMA, resulting in average increases over nontreated controls of 11.9-, 6.2- and 1.2-fold, respectively. This pretreatment was found to be synergistic when combined with centrifugation, having the same order of effectiveness, and resulting in 57-, 35- and 27-fold increases over nontreated controls. For Lipofectamine and DC-Chol-DOPE liposomes, the combined approach yielded 2.2- and 1.3-fold increases over untreated centrifuged samples. Individual colonies picked from colony-forming unit granulocyte-macrophage assays of infected CD34+ cells were screened for the presence of the transgene by polymerase chain reaction (PCR). Colonies from cells infected using centrifugation were positive 27% of the time, while the combined approach had positive colonies 31 and 50% of the time for DC-Chol and Lipofectamine, respectively. The addition of protamine sulfate to the liposome-supernatant mixture during pretreatment was found to be inhibitory. With increasing centrifugal force, the TE of cells infected with Lipofectamine pretreated and untreated supernatants increased proportionally. However, the TE of the cells infected with the pretreated supernatants was significantly higher than the TE of the cells infected with untreated supernatants at all points examined. The increase in TE associated with liposomal pretreatment of retroviral supernatants was not shown to be attributed to a nonreceptor-mediated pathway for viral entry into the cell.

Consistent numerical chromosome aberrations in congenital fibrosarcoma.

Cytogenetic analysis of a congenital fibrosarcoma of the volar forearm from a 2.5-month-old boy revealed a mosaic karyotype 46,XY/49,XY,+11,+17,+20. This pattern of specific trisomies provides additional support to the cytogenetic findings in five cases of congenital fibrosarcoma reported previously. Trisomy 11 appears to be characteristic of congenital fibrosarcoma with additional trisomies 8, 17, and 20 as common findings.