Transduction of Primitive Human Marrow and Cord Blood-Derived Hematopoietic Progenitor Cells With Adeno-Associated Virus Vectors by Saswati Chatterjee, Wei Li, Christie Ann Wong, Grace Fisher-Adams, Di Lu, Mausumee Guha, James A. Macer, Stephen J. Forman, and K.K. Wong Blood Volume 93(6):1882-1894 March 15, 1999 ©1999 by American Society of Hematology

Maps of AAV vectors used. Maps of AAV vectors used. Bold arrows denote the direction of transcription of each gene. The promoters, polyadenylation signals (PA), and the AAV ITR are shown. Light arrows denote the location of PCR primers. Primers RPSP and DLASP were used for amplification from DNA templates. Primers 1a and 1b were used for reverse transcription and amplification from RNA templates. Probes used for FISH analyses and Southern hybridization of RT-PCRs and dot blot analyses of vectors are designated. (A) vCWRHIVASVN encodes two transgenes: (1) an antisense RNA (A) complementary to the HIV LTR, including the TAR and the polyadenylation sequences under the transcriptional control of the RSV LTR; and (2) the neomycin phosphotransferase (NeoR) gene under the control of the SV40 early promoter. (B) vCWRHIVAPAP contains the antisense RNA gene cassette described in (B) in addition to the PLAP gene under the control of the PGK promoter. Saswati Chatterjee et al. Blood 1999;93:1882-1894 ©1999 by American Society of Hematology

Amplification of vCWRHIVAPAP sequences from individual LTC-IC colonies. Amplification of vCWRHIVAPAP sequences from individual LTC-IC colonies. CD34 cells were transduced with vCWRHIVAPAP at a functional MOI of 3 (particle MOI, 600) on day 0 and plated in long-term culture as described in Materials and Methods. Cells were harvested from stromal layers at designated time points, washed, and plated in methylcellulose with no G418 selection and colonies were plucked after 2 weeks. DNA was extracted and amplified for either the AAV vector using primers RPSP and DLASP or for β globin. β Globin served as a control for template integrity. The 418 HIVA band denotes the vector signal. The 268-bp Glo band denotes the β globin signal. Representative colonies from untransduced controls (Untd) are shown. Copy number controls are included in each analysis. For HIVA, the copy number corresponds to 12, 120, and 1,200 copies of the genome. For β globin, copy number controls show amplification from 80, 160, and 1,000 cells. (A) Week-5 LTC-ICs from donor TS. All colonies except one had intact DNA, albeit at varying quantities. The absence of a β globin signal from colony 9 indicated that the DNA template was inadequate. (B) Amplification of vCWRHIVAPAP sequences from individual week-8 LTC-IC colonies from donor TS. All colonies analyzed had intact DNA templates and 9 of 15 showed vector-specific signals. The standard curve is reversed in this experiment. (C) Amplification of vCWRHIVAPAP sequences of individual week-8 (transduced lanes 1 through 10) and week-10 (transduced lanes 11 through 20) LTC-IC colonies from donor HJ. Nine of 10 week-8 LTC-ICs had intact templates. Six of these showed vector-specific signals. All 10 week-10 colonies analyzed had intact DNA, and 6 of these were transduced. (D) Amplification of vCWRHIVAPAP sequences of individual week-5 and -10 LTC-IC colonies from donor FK. Colony 4 had intact albeit very low amounts of DNA as evidenced by a faint β globin signal on a prolonged radioautographic exposure. (E) Amplification of methylcellulose samples from between colonies in transduced LTC-IC wells. Ten separate samples of methylcellulose were amplified with primers RPSP and DLASP using PCR conditions identical to above. NT, no template; C, CWRHIVAPAP control (10 copies). This film was exposed for 36 hours longer than exposures in (A) through (D)‏ Saswati Chatterjee et al. Blood 1999;93:1882-1894 ©1999 by American Society of Hematology

FISH analysis of vCWRHIVAPAP transduced and untransduced cells. FISH analysis of vCWRHIVAPAP transduced and untransduced cells. CD34 were transduced with vCWRHIVAPAP at a functional MOI of 3 (particle MOI, 600) and placed in culture as described above. Suspension cells were harvested at designated time points for analysis. (A) A metaphase spread from an untransduced CD34 culture showing no hybridization of the vector-specific probe to cellular sequences. (B) Hybridization analysis of HIIC21, a G418-resistant clonal 293-based cell line derived after transduction with vCWRHIVASVN. This clone carries 1 copy of the vector genome per cell (28) and served as a positive control. (C, D, and E) Representative metaphases from vCWRHIVAPAP transduced CD34 cultures at 5 weeks posttransduction. Note that vector-specific signals are observed on both sister chromatids. Signals were not digitally enhanced in this analysis. (F) Representative interphase nuclei from a vCWRHIVAPAP transduced CD34 culture 3 weeks posttransduction. Three nuclei with three or more signals, two nuclei with two signals each, and three nuclei with no signals are seen. Saswati Chatterjee et al. Blood 1999;93:1882-1894 ©1999 by American Society of Hematology

Southern analysis of HIV antisense transcription from vCWRHIVASVN in transduced marrow and cord blood-derived hematopoietic progenitor cells after RT-PCR amplification. Southern analysis of HIV antisense transcription from vCWRHIVASVN in transduced marrow and cord blood-derived hematopoietic progenitor cells after RT-PCR amplification. (A) RNA was extracted from week-5 to -7 LTBMCs and antisense sequences were reverse transcribed and amplified using primers 1a and 1b. The amplified products were resolved on a 1.2% agarose gel, transferred to nitrocellulose, and hybridized with an RSV LTR and antisense-specific probe. HIIC21, containing 1 copy of integrated vector per cell, served as the positive control. The 530-bp antisense transcript-specific product is shown. +RT and −RT refer to the presence or absence, respectively, of reverse transcription before amplification. The absence of signals in these lanes indicates that the antisense signals were RNA-specific. ψ, RT-PCR analysis of cells (donor 20) exposed to a sham stock of vector prepared in the absence of AAV rep and cap genes. Untransduced (Untd) cells from each donor tested served as negative controls. Td, transduced cultures. (B) HIV LTR antisense transcription in LTC-ICs initiated with CD34+CD38− cord blood cells 8 weeks after transduction. A representative sample, CB6, was transduced with vCWRHIVAPAP at MOI of 3 (particle MOI, 600) and vCWRHIVASVN at MOI of 0.1 (particle MOI, 50). Cells were harvested from 6 week LTBMCs, washed, and placed in colony-forming assays. RNA was extracted from colonies after 3 weeks, reverse transcribed, and amplified with primers 1a and 1b. The 530-bp antisense product was evident only after reverse transcription. Saswati Chatterjee et al. Blood 1999;93:1882-1894 ©1999 by American Society of Hematology