Correction of murine Rag2 severe combined immunodeficiency by lentiviral gene therapy using a codon-optimized RAG2 therapeutic transgene.
Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2(-/-) mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2(-/-) mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands.
Niek P
NP
van Til
Helen
H
de Boer
Nomusa
N
Mashamba
Agnieszka
A
Wabik
Marshall
M
Huston
Trudi P
TP
Visser
Elena
E
Fontana
Pietro Luigi
PL
Poliani
Barbara
B
Cassani
Fang
F
Zhang
Adrian J
AJ
Thrasher
Anna
A
Villa
Gerard
G
Wagemaker
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D049109:Cell Proliferation; D046528:Chimerism; D002843:Chromatin; D003062:Codon / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D018628:Gene Dosage; D015321:Gene Rearrangement; D019674:Genes, T-Cell Receptor beta; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D016513:Mice, SCID; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D016511:Severe Combined Immunodeficiency / Q000235:genetics* / Q000628:therapy*; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D013601:T-Lymphocytes / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes
D002843:Chromatin; D003062:Codon; D004268:DNA-Binding Proteins; C499308:Rag2 protein, mouse