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, 4 (7), 775-80

T Lymphocytes With a Normal ADA Gene Accumulate After Transplantation of Transduced Autologous Umbilical Cord Blood CD34+ Cells in ADA-deficient SCID Neonates

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T Lymphocytes With a Normal ADA Gene Accumulate After Transplantation of Transduced Autologous Umbilical Cord Blood CD34+ Cells in ADA-deficient SCID Neonates

D B Kohn et al. Nat Med.

Abstract

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.

Figures

Fig. 1
Fig. 1
Purine metabolic parameters and immune function, a, PEG-ADA dosages. Dosages of PEG-ADA enzyme replacement therapy (U/kg per week) administered to subjects 1 (red), 2 (blue) and 3 (yellow), b, Plasma ADA levels. The levels of plasma adenosine deaminase enzymatic activity (●) measured at successive times are shown for subjects 1 (red), 2 (blue) and 3 (yellow), c, T-lymphocyte numbers. The absolute numbers of CD3+ T lymphocytes per mm3 of whole blood measured by FACS analysis are shown (●) for subjects 1 (red), 2 (blue) and 3 (yellow), d, T-lymphocyte blastogenesis. The proliferative responses of peripheral blood mononuclear cells to the T lymphocyte mitogen PHA (●) and the antigen tetanus toxoid (X) measured at successive times are shown for subjects 1 (red), 2 (blue) and 3 (yellow).
Fig. 2
Fig. 2
Frequency of gene-containing leukocytes. The frequency of cells containing the LASN vector sequences as measured by semi-quantitative PCR in PBMC fractions (●), granulocytic fractions (X), and FACS-sorted CD3+ T lymphocytes (T), CD13+/CD14+ monocytic cells (M) and CD19+ B lymphocytes (B) at successive times are shown for subjects 1 (a), 2 (b) and 3 (c).
Fig. 3
Fig. 3
RT-PCR analysis of vector expression in leukocytes. The products were generated by reverse transcription of RNA using a primer which was antisense to the ADA cDNA, followed by PCR amplification of cDNA using primers from the LASN vector. Samples were from peripheral blood leukocytes obtained from subject 1 four-and-a-half years after gene transfer. Samples were analyzed in duplicate, with (+) or without (−) reverse transcriptase, to control for DNA contamination. Leukocyte samples include PBMC (PBMC) and PHA-stimulated PBMC (PHA). Portions of each RNA sample were also reverse transcribed and amplified with primers to β-actin (beta-actin), to verify that equivalent amounts of RNA were assayed.

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