Supplementary Information. Novel lentiviral vectors with mutated reverse transcriptase for mrna delivery of TALE nucleases

Transcription

1 Supplementary Information Novel lentiviral vectors with mutated reverse transcriptase for mrna delivery of TALE nucleases Ulrike Mock 1, Kristoffer Riecken 1, Belinda Berdien 1, Waseem Qasim 2, Emma Chan 2, Toni Cathomen 3,4, Boris Fehse 1 1 Research Dept. Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany. 2 Molecular Immunology Unit, UCL Institute of Child Health, London, WC1N 1EH, United Kingdom 3 Institute of Cell and Gene Therapy and 4 Center for Chronic Immunodeficiency, University Medical Center Freiburg, Engesserstr. 4, Freiburg, Germany Supplementary information Mock et al. Page 1

2 Supplementary Figure 1 a A 2658 bp A SFFV N-term repeat modules (1-18) linker FokI b B 3765 bp B Supplementary Figure 1. Recombination events in TALEN-encoding LVV (a) Schematic representation of PCR-design. Primer-set A was used for proviral DNA (u40/41; u42/43) and B for reverse transcribed viral mrna (p07/u37). (b) Recombination in TALEN-encoding LVV determined by sequencing of integrated proviruses in transduced cells (bulk-culture). DNA of a transduced 293T-bulk culture was isolated, proviral DNA was PCR-amplified, subcloned and sequenced. Recombination pattern (dark grey) for either left arm (TALEN L) or right arm (TALEN R) is depicted. Light grey blocks represent monomers that could not be identified unambiguously. In total 15 clones were analyzed, 15/15 showed recombination, 15/15 were in frame. In 10/15 clones one, in 4/15 at least two recombination events were detectable. Clone #2 (TALEN L) could not be assigned definitely. Supplementary information Mock et al. Page 2

3 Supplementary Figure % egfp-positive hours post transduction Supplementary Figure 2. Kinetics of egfp-expression post transduction with NRTLV. Transduction of 293T cells was performed with NRTLV-iG2, and percentages of egfp-positive cells were measured at indicated time points by flow cytometry. Summarised data for 3 independently produced vector preparations is shown. Error bars indicate SEM. Supplementary information Mock et al. Page 3

4 Supplementary Figure Mean Fluorescence Intensity, relative to DMSO control [%] DMSO control 5µg/mL 5ug/mL 50ug/mL 50µg/mL 100µg/mL 100ug/mL Concentration of Cycloheximide Supplementary Figure 3. mrna transfer is the main mechanism of non-reverse transcribable lentiviral vectors (NRTLV). 293T cells were transduced with NRTLV LeGO-iG2 two hours after addition of the translation-inhibiting compound cycloheximide. Mean fluorescence intensity of egfp was measured by flow cytometry after incubation at 32 C for 24 hours and corrected for autofluorescence. The DMSO control reflects both egfp-protein transfer and de-novo translated egfp, whereas in the presence of cycloheximide egfp fluorescence is obviously due to protein transfer. MFI values obtained for DMSO controls were set to 100%, and relative values were defined for the three experimental groups. Error bars indicate SD for n=3. Supplementary information Mock et al. Page 4

5 Supplementary Figure 4 a CMV I : LeGO-iTALEN-L-iG2-BGH-p(A) / LeGO-iTALEN-R-iG2-BGH-p(A) Ψ RRE TALEN egfp BGH-p(A) II : LeGO-iTALEN-L-iG2--BGH-p(A) / LeGO-iTALEN-R-iG2--BGH-p(A) CMV Ψ RRE TALEN egfp BGH-p(A) III : LeGO-iTALEN-L-iG2-SV40-p(A) / LeGO-iTALEN-R-iG2-SV40-p(A) CMV Ψ RRE TALEN egfp SV40-p(A) IV : LeGO-iTALEN-L-iG2--SV40-p(A) / LeGO-iTALEN-R-iG2--SV40-p(A) CMV Ψ RRE TALEN egfp SV40-p(A) b Fold increase (MFI egfp) * * ** ** 0.0 italen-ig2- italen- ig2- SV40p(A) italen- ig2- BGHp(A) italen- ig2- - SV40p(A) italen- ig2- - BGHp(A) Supplementary Figure 4. Improved expression of TALEN constructs with different p(a)-signals. (a) Schematic vector design of I: LeGO-iTALEN-iG2-BGH-p(A) II: LeGO-iTALEN-iG2--BGH-p(A) III: LeGO-iTALEN-iG2-SV40-p(A) IV: LeGO-iTALEN-L-iG2--SV40-p(A). 3 rd generation LVV derived from LeGO-system (Weber et al.). CMV = CMV-ie promoter; ( U3), R, U5 = elements of SIN-LTR, self-inactivating long terminal repeat; Ψ = Psi, packaging signal; RRE = Rev response element; SFFV = promoter of spleen focus-forming virus; = Woodchuck hepatitis virus posttranscriptional regulatory element; = internal ribosome entry site; egfp = enhanced green fluorescent protein; p(a) = polyadenylation signal; BGH = bovine growth hormone; SV40 = simian virus 40. (b) Transduction of 293T cells was performed with non-concentrated preparations of NRTLV encoding TALEN and egfp. Expression was assessed based on mean fluorescence intensities (MFIs) for egfp measured 48h post transduction by flow cytometry and normalized to italen-ig2-. The highest increase in MFI was observed for the two lentiviral vector constructs with internal, exogenous p(a)-signals from the bovine growth hormone (BGH) or the simian virus 40 (SV40) directly following the signal [italen-ig2--bghp(a) and italen-ig2--sv40p(a)]. Introduction of the respective p(a)-signals upstream of the led to a slightly lower, but still significant increase of expression [italen-ig2-bghp(a); italenig2-sv40p(a)]. All constructs are schematically depicted in Supplementary Figure 2d. Means for 3 independently produced vector preparations, each measured in duplicates, are shown. Error bars indicate SD (* p < 0.01; ** p < 0.005). = Woodchuck hepatitis virus posttranscriptional regulatory element; i = internal ribosome entry site (). Supplementary information Mock et al. Page 5

6 Supplementary Figure % egfp-positive (d2) % CCR5 knockout MOI per vector 0 Supplementary Figure 5. Correlation between egfp-expression and CCR5 knockout after transduction with NRTLV. Clonal CCR5+ 293T cells were co-transduced with increasing MOIs of concentrated NRTLVs delivering the CCR5-specific italen-constructs with improved /BGH-p(A)-containing vectors. egfpexpression was measured on d2 post transduction (left axis, light grey bars) and CCR5 knockout on d5 post transduction (right axis, dark grey bars) by flow cytometry. Data shown for n = 3. NRTLV = non-reverse transcribable lentiviral vector; i = internal ribosome entry site (); = Woodchuck hepatitis virus posttranscriptional regulatory element; BGH = bovine growth hormone. Supplementary information Mock et al. Page 6

7 Supplementary Figure 6 untransduced MOI 1250 MOI 2500 egfp untransduced MOI 1250 MOI 2500 CCR5-PerCP-Cy5.5 Supplementary Figure 6. Marking but no knock-out with CCR5-specific TALEN delivered by NRTLV. Primary T cells from different donors were activated and transduced with either MOI 1250 or MOI 2500 per TALEN-arm as NRTLV on day 3 post activation. egfp-expression was measured on d2 post transduction (upper panel) and CCR5 knockout on d5 post-transduction (lower panel) by flow cytometry. Data shown for 1 representative donor out of 3. Supplementary information Mock et al. Page 7

8 Supplementary Table 1 Oligonucleotide sequences Number Sequence (5-3 ) Target u8 u9 u28 u29 u30 u31 u40 u41 u42 u43 p74 u37 u44 u45 u46 u47 AAGATGGATTATCAAGTGTCAAGTCC GATGATTCCTGGGAGAGACGC AGTCCTCCGACAGACTGAGT TGCAGGTCGACTCTAGAGTC TGCATGCATGGCGCAAT AACTGGGCAACAATGCTCTC ATCTACGCACGCTCGGCTA CTGCTTGGCCAATTGGCAGAT AAGGTTCGTTCGACAGTGG CACACCGTAATCAATAGGAGATC ATCAGCCTGCTTCTCGCTT CATACGGGAAGCAATAGCATGATAC ACAATGAGACACCA GATATGTCCATTGGCCTTGCCC TACATACAACACCACCATGTAT CATGGGCTACCCTTACGACGTGCCTGACTACGCCTCTAG ACCCAAGAAAAAGCGGAAAGTGGGCATCCACG hccr5-f hccr5-r TALEN-seq F1 TALEN-seq R1 TALEN-seq F2 TALEN-seq R2 TALEN-F 1st PCR TALEN-R 1st PCR TALEN-F 2nd PCR TALEN-R 2nd PCR SFFV-F -R pmdl_gp_rre_r pmdl_gp_rre_f pmdl_gp_rre_mut italen oligo sense u48 u58 u59 u60 u61 u62 u63 CTAGCGTGGATGCCCACTTTCCGCTTTTTCTTGGGTCTA GAGGCGTAGTCAGGCACGTCGTAAGGGTAGCC GCTGTACAAGTAACTGTGCCTTCTAG GCTGTACAGCCATAGAGCCCAC GCTGTACAAGTAAAACTTGTTTATTGCAGC GCTGTACACAGACATGATAAGATACATTGATGAGT GGTCTAGACCAAAGAAGAAGCGGAAGG TAGCGGCCGCTTATGAGCGGAAATTG italen oligo antisense BGH-p(A)-F BGH-p(A)-R SV40-p(A)-F SV40-p(A)-R italen-tcrα2-f italen-tcrα2-r Supplementary information Mock et al. Page 8