T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates

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.

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

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

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.