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Blood, Vol. 113, Issue 21, 5104-5110, May 21, 2009
Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection
Blood Throm et al.
113: 5104
Supplemental materials for: Throm et al
Supplemental Methods Plasmid Constructions. To make the HIV gagpol vector plasmid (pSFG-IC-HIVgp-Ppac2) we removed the tetracycline dependent promoter driven luciferase from pSFG tcLuc ECT31 and replaced it with a CMV enhancer/promoter driven codon optimized HIV NL4-3 gagpol gene, and a PGK promoter driven puromycin resistance gene derived from pMSCVpac.2 The same codon optimized HIV gagpol was inserted into pCAGGS3 to make pCAGG-HIVgpco. The HIV rev and tat genes were also based on the NL4-3 strain sequence and were inserted directly in place of luciferase in pSFG tcLuc ECT3, to make pSFGtc-revco and pSFGtc-tatco. The VSV-G coding sequence from pMD.G4 was also inserted similarly into this vector plasmid to make pSFGtc-VSVG. pSFG-tTA is described in Lindemann, et al.1 pHDM.G is a high copy derivative of pMD.G.4 pTL20-MSCV-GFP was constructed by replacing the SpeI-NcoI fragment containing the CMV enhancer sequences in pCL20c-MSCV-GFP5 with a similarly ended, 322 bp PCR fragment containing the 7 tet operator cassette amplified from pSFG tcLuc ECT3. pTL20i4r2-MSCV-YFP is a further modified vector plasmid containing a 400 bp chicken HS4 insulator fragment6 inserted into the 3′ U3 at the BbsI site, an additional Sfi I site upstream of the vector backbone (via insertion of the synthetic sequence GGCCGCCTCGGCC into the unique PmeI site), for generation of fragments with non-palindromic overhangs, and with YFP replacing GFP. The plasmid pLT-PGK-ble was constructed by ligating 1) a SmaI-BamHI fragment containing the murine PGK promoter (ranging from −393 to +73 relative to the start of transcription) from HRST-IPGK-GFP-WS,7 2) a BamHI-ApoI fragment containing the bleomycin resistance gene (PCR amplified from pJ6-omega-Bleo), 3) an ApoI-SpeI fragment containing the SV40 late polyadenylation sequence from pscAAV-LP1-hFIXco,8 and 4) an SpeI-EcoRV digested cloning vector LITMUS29 (NEB). pLT-PGK-ble-v3 additionally contains PflM I sites (CCAGCCTTT) inserted at the EcoRI and SnaBI sites to flank the PGK-ble cassette for producing fragments for directional concatamerization. To make concatemeric DNA for transfections,25–100 µg of vector and PGK-ble plasmids were independently digested with the enzymes indicated in the methods, and gel purified. Purified DNA was quantitated, and mixed in a 25:1 vector:PGK-ble molar ratio while maximizing the total DNA concentration, and ligated using the NEB Quick Ligation kit. After ligation, DNA was purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. Between 3–15 µg of DNA was transfected onto one 10 cm dish of GPRG cells. The CL20i4r-EF1a-hgcOPT plasmid was constructed by replacing the BstBI-NotI fragment of CL20i4r-MSCV-GFP with the sequence indicated in Fig. S4. Western blot analysis. Cells were lysed in ice cold lysis buffer (50mM Tris pH8.0, 150mM NaCl, 1mM EDTA and 1% Triton X-100 mixed with one Complete, Mini tablet, Roche). Cell lysates were quantitated using the Bradford microassay, and fifty micrograms of total protein were analyzed per sample, while for supernatants 34 µl was used. HIV p24 and VSV-G expression were monitored using monoclonal antibody 39/6.14 (Abcam) and a sheep polyclonal antibody (Abcam), respectively. A sheep polyclonal antibody (Novus Biologicals) was used to detect HIV Rev expression. After probing with the appropriate horseradish peroxidase-tagged secondary antibody, signal was visualized using enhanced chemiluminescence detection reagents (GE Healthcare). Real-time quantitative PCR. Genomic DNA was purified from producer cells using the Gentra Puregene Cell kit (Qiagen), and 2 ng used for quantitative PCR reactions containing primers with sequence 5′ ACTTGAAAGCGAAAGGGAAAC-3′ and 5′- CACCCATCTCTCTCCTTCTAGCC-3′, and probe 5′FAM AGCTCTCTCGACGCAGGACTCGGC-3′, and the MyIQ Supermix (Bio-Rad). 45 cycles of (94°C 15 seconds, 60°C 15 seconds) were performed on a MyIQ Thermal Cycler (Bio-Rad), using linearized vector plasmid DNA as a quantitative standard. Statistics. The statistical significance of the difference in titers produced by clones generated by the two methods was determined using the Wilcoxon-Mann-Whitney test. Bioreactor production of HIV vector from stable producer cell line. The Wave bioreactor 20/50 system was assembled and operated according to manufacturer’s recommendations. A Wave cellbag 10L with 100g of FibraCel disks was inoculated at 2 × 105 cell/ml in 2500 ml of D10 + 2mM Glutamax (Invitrogen) + 1 ng/ml doxycycline (Clontech). The rocking speed of the bioreactor was set at 10 rocks per minute at an angle of 7 degrees. The culture was maintained at 37°C, 5% CO2 and pH of 7.5. On Day 4, the culture was refed with 5 L of D10 plus 1 ng/ml of doxycycline. On day 7, the FibraCel disks were washed twice with 2.5 L of PBS and once with 2.5 L of DMEM. The culture was fed with 5 liters D10 + 2mM Glutamax. After 24 hrs, the conditioned supernatant was harvested and the culture was feed with 5 liters D10 + 2mM Glutamax. The media was supplemented with glucose to maintain the glucose level >2.0 g/L. The conditioned media was filtered using either Opticap XL2 Durapore 0.45 µm filters (Millipore) or Opticap XL10 1.2 µm prefilter and Opticap XL10 Durapore 0.45 µm filter. The harvesting and filtration of the conditioned media and the refeeding of the Wave culture was repeated every 24 hrs for the next 7 days. The supernatants with the five highest titers were pooled together, and 13.8 L of this material was concentrated using a Pellicon-2 mini filter, 0.1M2 (Millipore) with a 500 kDa cutoff was used to concentrate the supernatant 18 fold. The concentrated supernatant was filtered using an Opticap XL2 Durapore 0.45 µm filter before vialing and storage at −80°C. Southern blot analysis. Genomic DNA was isolated from cells using the Gentra Puregene Cell kit (Qiagen) as per the manufacturer’s instructions, and five micrograms of DNA was digested using EcoRI, for Concatamer DNA analysis (Fig. 4b), or BglII, for analysis of HeLa cells transduced with lentiviral vectors (Fig. S2), or SbfI, for analysis of ED7R cells transduced with EF1a-hgcOPT (Fig. 6c). Samples were electrophoresed through a 0.8% agarose gel, transferred to a Zeta-Probe GT membrane (Bio-Rad), and probed with a 32P labeled fragment containing the RRE, and imaged using storage phosphor screens. REFERENCES 1. Lindemann,D., Patriquin,E., Feng,S., & Mulligan,R.C. Versatile retrovirus vector systems for regulated gene expression in vitro and in vivo. Mol. Med. 3, 466–476 (1997).
2. Hawley,R.G., Lieu,F.H., Fong,A.Z., & Hawley,T.S. Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1, 136–138 (1994).
3. Nitta,Y. et al. Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice. Hum. Gene Ther. 9, 1701–1707 (1998).
4. Ory,D.S., Neugeboren,B.A., & Mulligan,R.C. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. U. S. A. 93, 11400–11406 (1996).
5. Hanawa H et al. Efficient Gene Transfer into Rhesus Repopulating Hematopoietic Stem Cells using a Simian Immunodeficiency Virus-Based Lentiviral Vector System. Blood Online, (2004).
6. Aker,M. et al. Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. Hum. Gene Ther. 18, 333–343 (2007).
7. Mostoslavsky,G. et al. Efficiency of transduction of highly purified murine hematopoietic stem cells by lentiviral and oncoretroviral vectors under conditions of minimal in vitro manipulation. Mol. Ther. 11, 932–940 (2005).
8. Nathwani,A.C. et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood. 107, 2653–2661 (2006).
Files in this Data Supplement:
- Figure S1. Western blots of cell lysates and supernatants for packaging cell lines and 293T cells (JPG, 66.8 KB)
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Identified proteins are indicated at right, and the migration of size markers, in kDa, at left. 293T cells are shown with (+) and without (−) transient transfection of plasmids expressing the relevant protein. (A) GP, GPR, and GPRT cell lysates and (B) supernatants probed with antibody specific for HIV p24, which is expressed constitutively. (C) GPR and (D) GPRT cell lysates, probed with antibody specific for HIV Rev, at various times after induction by removing doxycycline. (E) GPRG cell lysates probed with an antibody specific for VSV-G, at various times after induction by removing doxycycline.
- Figure S2. Southern blot analysis of BglII digested HeLa cell genomic DNA after transduction with producer cell supernatants (JPG, 24.3 KB)
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(A) Image of Southern blot. Lane 1, cells transduced with 0.5 ml tangential filtration concentrated product from GPRG-TL20-GFP in bioreactor. Lane 2, cells transduced with control vector produced by transient transfection, and yielding 30% GFP+ cells, as determined by flow cytometry. Lane 3, cells transduced with 0.5 ml unprocessed supernatant from selected, directional concatamer transfected GPRG cells. (B) Map of vector genomes, in the proviral form, with expected BglII fragment size, and the location of the probe indicated. Note that the expected fragment size is not affected by the presence of the insulator within the LTR. XFP represents GFP for the GPRG-TL20-GFP vector product, and YFP for the directionally ligated concatemer produced vector product.
- Figure S3. Ethidium bromide stained agarose gel of concatemeric DNA digested with EcoRI (JPG, 36.7 KB)
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Designations from diagrams in Fig. 5A are indicated at center, in addition to sizes from markers, in kb, at sides.
- Figure S4. Fully annotated sequence of the EF1a-hgcOPT fragment used in the clinical vector construction (JPG, 439 KB)
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Sequence of the fragment inserted into CL20i4r-MSCV-GFP as described in supplemental methods is shown with annotations below the sequence. The EF1alpha promoter is indicated in green and yellow for sequences upstream and downstream, respectively, of the transcription start site. The IL2RG coding sequence and one letter amino acid translation is also indicated.
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