Supplemental Methods Chemicals and Reagents Vector production BLAM-Vpr assay

Transcription

1 Supplemental Methods Chemicals and Reagents Rapamycin and bafilomycin A1 were obtained from Sigma-Aldrich. Chloroquine was obtained from Life Technologies as part of an LC3B antibody kit. Torin 1 was obtained from Tocris Bioscience. Tatbeclin 1 peptide was obtained from Life Technologies. FK56 was obtained from Invivogen. Vector production Third generation CG-UbiC-EGFP LV was produced, concentrated, and titered as previously described 1. BLAM-Vpr-LV was produced by co-transfection with a BLAM-Vpr expressing plasmid (previously described 2 ) at a 1:3 ratio to CG-UbiC-EGFP. GFP-Vpr-LV was produced by co-transfection with a GFP- Vpr expression plasmid (previously described 3 ) at a 1:3 ratio and substituting CG-UbiC-EGFP for mcherry-expressing CG-UbiC-mCherry. The Lassa envelope for pseudotyping LV 4 was a kind gift from Dr. Michael Oldstone, The Scripps Research Institute, La Jolla, CA. LV production, concentration, and titer was previously described 1. Second generation RRL-MND-GFP LV 5 was produced by cotransfection of RRL-MND-GFP (6 μg), pspax2 (3 μg) and MD2G (1.5 μg) per plate into HEK 293T cells by PEI (Polysciences, Inc.) mediated transfection. Integration-deficient RRL-MND-GFP was produced by using pspax2 D64V instead of pspax2. LV supernatant was harvested 48 hours post transfection and concentrated by overnight centrifugation at 85 xg. LV Titer was determined by transduction of Nalm6 cells. BLAM-Vpr assay Human cord blood CD34 + cells were pre-treated for 6 hours with DMSO or rapamycin, then transduced with BLAM-Vpr-LV (MOI=25) for another 6 hours in the presence of the drugs. Cells were then washed and resuspended in 125 μl loading medium (IMDM containing 2% BIT95, no antibiotics). BLAM substrate loading was carried out according to manufacturer s protocol (LiveBlazer FRET B/G loading kit with CCF4-AM, Life Technologies) for 8 hours. Cells were fixed in 1.2% paraformaldehyde, and the percentage of cells exhibiting blue fluorescence, indicative of cleaved substrate, were read on a BD LSRII equipped with a UV laser. Quantification of HIV-1 reverse transcription products and LV copy number determination Human cord blood CD34 + cells (2-3x1 5 per sample) were transduced (MOI=25) in the presence of DMSO or 1-2 μg/ml rapamycin. At 6, 12, and 24 hours after start of transduction, cells were trypsin treated to remove surface-bound LV, pelleted, and frozen. Total DNA was extracted using QIAamp DNA mini kit (Qiagen), and treated with DpnI (New England BioLabs) for 2 hours to eliminate residual packaging plasmid DNA if present. Quantitative PCR was carried out according to a previously published protocol 6. Reactions were run on a Roche LightCycler 48, using LightCycler 48 Probes Master (Roche). Determination of LV copy number was accomplished as previously described 7. All primers are available on request.

2 Cell viability and growth, and colony forming unit assessment Human cord blood CD34 + cells were transduced with CG-UbiC-EGFP or PBS only, in the presence of no drug, DMSO only, or indicated concentrations of rapamycin. Cells were then washed and seeded, in duplicate or triplicate, in serum-containing expansion medium for up to 14 days. At the desired times aliquots of transduced cells were resuspended in FACS buffer containing 1 μg/ml propidium iodide, and viability was determined via flow cytometry as the percentage of propidium iodide exclusion after pre-gating to remove debris. For growth assessment, live cells from each sample, in duplicate or triplicate, were counted on a hemacytometer, and cell numbers were normalized to the no-drug, mocktransduced sample. Colony-forming unit (CFU) assessment of CD34 + cells and EGFP assessment was previously described 8. Methocult H434 media for CFU assessment was purchased from StemCell Technologies. Confocal microscopy Human cord blood CD34 + cells (4x1 5 cells per condition) were transduced with EGFP-Vpr-LV (MOI=25) in the presence of 1 μm chloroquine, 25μM Tat-beclin 1, 1 μg/ml rapamycin, DMSO only, or no drug. Cells were then washed, fixed in 3.7% paraformaldehyde, permeabilized in.2% Triton X-1, and blocked in 5% goat serum. Cells were stained with 1:2 diluted anti-lc3b (Sigma Aldrich) at room temperature for 1 hour, followed by 1:25 diluted Alexa 594 goat anti-rabbit IgG (Life Technologies) at room temperature for 45 minutes, and nuclear staining using Hoechst (Life Technologies) at room temperature for 1 minutes. Cells were attached to microscope slides using a Cyto-spin at 4 rpm for 7 minutes, and mounted in ProLong Gold Anti-Fade mounting medium (Life Technologies). Images were acquired at room temperature on a BioRad Zeiss 21 confocal microscope using a 6x oil-immersion objective lens (NA=1.4), Nikon TE2-U camera, and BioRad LaserSharp 2 software. Images were processed using Fiji 9. Cell cycle assays Human cord blood CD34 + cells (6x1 5 cells per condition) were treated with DMSO or 1 μg/ml rapamycin for 6 or 12 hours. Cells were then resuspended in ice-cold PBS at 1x1 6 cells/ml, and fixed by adding 1 ml ice-cold 7% ethanol, immediately vortexing, and storing at -2 C for at least 24 hours. Fixed cells were resuspended in ice-cold HBSS with Ca 2+ /Mg 2+ at 1x1 6 cells/ml, and stained by adding an equal volume of ice-cold staining solution (HBSS with 4 μg/ml Pyronin-Y (Sigma Aldrich) and 2 μg/ml Hoechst (Life Technologies) and incubating on ice for 2 minutes. Fluorescence was immediately read on a BD LSRII. LV binding assay Human cord blood CD34 + cells were treated with CG-UbiC-EGFP (MOI=25) at 4 C in the presence of DMSO or 1 μg/ml rapamycin. After 2 hours, cells were washed three times with PBS to remove unbound LV. 6-8x1 4 cells from each sample were lysed in.5% Triton X-1. HIV p24 was quantified by the Center for AIDS Research at the University of California, San Diego.

3 Flow cytometry For immunophenotyping of human cells from cord blood or humanized NSG mice, anti-human CD45, CD3, CD33, CD38, CD4, CD8, CD14, and CD19 antibodies conjugated with appropriate fluorophores (BD Biosciences) were used. For immunophenotyping of cells from mouse-to-mouse transplants, antimouse CD45.1, CD45.2, CD3, CD4, CD8, CD18, GR-1, B22, CD11c, and CD11b antibodies (Ebiosciences, Biolengend and BD Biosciences) conjugated with appropriate fluorophores were used. For intracellular staining of p21/waf1/cip1, human cord blood CD34 + cells were fixed in 3.7% paraformaldehyde, permeabilized in.2% Triton X-1, and blocked in 5% goat serum. Cells were stained with 1:1 diluted anti-p21 clone CP74 (Sigma-Aldrich) at room temperature for 15 minutes, followed by 1:1 diluted Alexa 488 goat-anti-mouse IgG (Life Technologies) at room temperature for 15 minutes. For staining of surface LDLR and α-dystroglycan, human cord blood CD34 + cells were blocked in 5% goat serum. Cells were stained with 1:1 diluted anti-α-dystroglycan clone IIH6C4, or 1:1 diluted anti-ldlr clone C7 (both from Millipore) at room temperature for 15 minutes, followed by 1:1 diluted Alexa 488 goat-anti-mouse IgG (Life Technologies) at room temperature for 15 minutes. Fluorescence was read on a BD LSRII. Beclin heterozygous mice Bone marrow from Beclin heterozygous and littermate mice were kindly provided by Dr. Lenny Shultz, The Jackson Laboratory, Bar Harbor. ME. Loss of Beclin results in an embryonic lethal, whereas Beclin heterozygous mice survive and been shown to be deficient in autophagy mechanisms 1. RIS analysis Retrovirus integration site amplification, detection, and processing were carried out as previously described with the exception that random shearing was accomplished by adaptive focused acoustics technology 11. Processing and genomic mapping of retrovirus integration sites was carried out as previously described 11 with the following exceptions: valid integration sites were scored after locating retrovirus LTR, intervening mouse genomic DNA and linker cassette sequence. The resulting junction sequences were aligned to the December 211 (GRCm38/mm1) assembly of the mouse genome with a stand-alone version of BLAT 12 that generates a BLAST alignment score. All primers are available on request. To generate the whole genome view (Figure 6C), we used the scatterplot feature available from Circos ( 13. To analyze insertion events with respect to genic regions, integration sites for each mouse in the same treatment group (rapamycin or control) (Table 1) were combined and then analyzed using the QuickMap tool available from the Gene Therapy Safety Program 14. Intraosseous (IO) infusion of LVs into mice IO infusion procedure has been described previously 15,16. Briefly, a 27-gauge needle was carefully inserted into the right tibia through the joint of an anesthetized mouse. Twenty μl of RRL-MND-GFP ( TU/mouse) with or without rapamycin (25 μg/kg/mouse) loaded in a 1 μl Hamilton syringe connected via a 15 cm sterilized ultra-thin tubing to the needle was slowly infused into the tibia.

4 The infusion speed (1 μl/min) was precisely controlled by a syringe pump (NE-11, New Era Pump Systems). On day 62, the bone marrow cells were collected by flushing tibias and femurs, then EGFP expression in Lin - Sca-1 + C-Kit + HSCs was analyzed by flow cytometry. Statistical evaluation A parametric two- tailed Student s T test was used for statistical evaluation, unless otherwise noted in the figure legend. Throughout all figures lines represent group mean and error bars represent S.D. * denotes statistical significance of p<.5, ** denotes p<.1, *** denotes p<.1, and **** denotes p<.1.

5 Supplemental References 1. Swan CH, Buhler B, Steinberger P, Tschan MP, Barbas CF, 3rd, Torbett BE. T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther. 26;13(2): Tobiume M, Lineberger JE, Lundquist CA, Miller MD, Aiken C. Nef does not affect the efficiency of human immunodeficiency virus type 1 fusion with target cells. J Virol. 23;77(19): McDonald D, Vodicka MA, Lucero G, et al. Visualization of the intracellular behavior of HIV in living cells. Journal of Cell Biology. 22;159(3): Shimojima M, Ikeda Y, Kawaoka Y. The mechanism of Axl-mediated Ebola virus infection. J Infect Dis. 27;196 Suppl 2:S Zufferey R, Dull T, Mandel RJ, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol. 1998;72(12): Mbisa JL, Delviks-Frankenberry KA, Thomas JA, Gorelick RJ, Pathak VK. Real-time PCR analysis of HIV-1 replication post-entry events. Methods Mol Biol. 29;485: Astrakhan A, Sather BD, Ryu BY, et al. Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome. Blood. 212;119(19): Miyoshi H, Smith KA, Mosier DE, Verma IM, Torbett BE. Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science. 1999;283(542): Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biologicalimage analysis. Nat Methods. 212;9(7): Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest. 23;112(12): Adair JE, Beard BC, Trobridge GD, et al. Extended survival of glioblastoma patients after chemoprotective HSC gene therapy. Sci Transl Med. 212;4: Kent WJ. BLAT--the BLAST-like alignment tool. Genome Res. 22;12(4): Krzywinski M, Schein J, Birol I, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 29;19(9): Appelt JU, Giordano FA, Ecker M, et al. QuickMap: a public tool for large-scale gene therapy vector insertion site mapping and analysis. Gene Ther. 29;16(7): McCauslin CS, Wine J, Cheng L, et al. In vivo retroviral gene transfer by direct intrafemoral injection results in correction of the SCID phenotype in Jak3 knock-out animals. Blood. 23;12(3): Worsham DN, Schuesler T, von Kalle C, Pan D. In vivo gene transfer into adult stem cells in unconditioned mice by in situ delivery of a lentiviral vector. Mol Ther. 26;14(4):

6 17. Drake KR, Kang M, Kenworthy AK. Nucleocytoplasmic distribution and dynamics of the autophagosome marker EGFP-LC3. PLoS One. 21;5(3):e Chatterji U, Bobardt MD, Stanfield R, et al. Naturally occurring capsid substitutions render HIV- 1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in owl monkey cells. Journal of Biological Chemistry. 25;28(48):

7 Supplemental Table 1. Analysis of LV insertion profile using QuickMap Genomic Locations of Integration Events Observed Random Dataset (1E+6 random genomic positions) Frequency of Insertion Events Control Dataset (DMSO-treated; 124 non-redundant IS) Experimental Dataset (Rapamycin-treated; 31 non-redundant IS) Within Genes 39% 62.1% 58.5% Within Cancer Genes 1.4% 3.2% 2% 5 kb from Gene TSS 1.2% 12.1% 12% 5 kb from Cancer Gene TSS.2%.8%.3% 5 kb from Gene TSS 63.9% 82.2% 73.8% 5 kb from Cancer Gene TSS 1.9% 4.% 2.3% 25 kb from Gene TSS 152% 176.6% 159.1% 25 kb from Cancer Gene TSS 1% 1.5% 8.3% TSS: Transcription start sites.

8 Supplemental Table 2. Top 3 clones identified by capture frequency analysis in mouse bone marrow Lin - cells Treatment Group Control Group Rapa-treated Group Mouse No Top 3 Clones Identified (Descending Order) % % Chromosome: Frequency Contribution Position within RIS to Lin - BM Nearest TSS (Distance in kb) pool cells 17: Pacrg (51.2) 14: E6Rik (212.) 7: Ntrk3 (79.5) 15: Trps1 (77.7) X: Maged2 (24.3) 2: Nr4a2 (775.8) 4: Pik3cd (25.7) 7: Btbd1 (28.1) 16: Cb1b (37.8) 3: Snx16 (391.8) 5: Stim2 (91.5) 15: Cdh9 (395.7) 1: POC1B (55.6) X: Mageb18 (133.5) 17: Haao (1.8) 11: Pld6 (58.7) 8: Purg (49.5) 5: Arap2 (11.2) 19: Sorcs1 (36.8) 6: Moxd2 (82.6) 6: C4Rik (35.3) 1: Cdc73 (851.7) 17: Rasgrp3 (59.3) 11: Vrk2 (59.7) 15: Pkhd1l1 (18.4) 15: Kcnv1 (596.1) 14: Oxsm (44.2) The product + Lin - cells as determined by flow cytometry and the % frequency within the RIS pool. Nearest RefSeq Gene. TSS: transcription start site

9 Supplemental Figures A Viability Day 4 Day 6-7 Day 1-12 % Viability LV + LV 85 no drug DMSO only Rapa (ug/ml) 2 85 no drug DMSO only no drug DMSO only B Cell count Normalized cell count no drug DMSO only Rapa (ug/ml) no drug DMSO only no drug DMSO only LV + LV C FSC characteristics 12 hours 11 days FSC Supplemental Figure 1. Viability and growth of rapamycin-treated CB CD34 + cells. CD34 + cells were pre-stimulated and either transduced (open green squares) or not (filled red circles) with CG- UbiC-EGFP, MOI=25, using the transduction protocol presented in Figure 1A and Methods. Cells were maintained in culture as discussed in Methods. No drug indicates cultures without DMSO or rapamycin. (A) At indicated days post the initiation of transduction, cell viability was determined by propidium iodide exclusion via flow cytometry. (B) At indicated days post the initiation of

10 transduction, cell numbers were counted using a hemacytometer, and normalized to the non- transduced, non- drug treated sample. (C) Forward scatter (FSC) profile of transduced CD34 + cells, 12 hours and 11 days post transduction. Black, DMSO only; red, 1 μg/ml rapamycin. For viability and cell counts, data are pooled from 2-3 independent experiments, each with different CB donors. Lines represent group mean and error bars represent S.D.

11 A CFU plating efficiency B Colony type distribution plating efficiency (%) n.s. 5 * % of total colonies 1 5 CFU-GEMM CFU-M CFU-GM BFU-E 1 2 Rapa (ug/ml) 1 2 Rapa (ug/ml) C 5 Percentage of GEMM colonies n.s. n.s. % of total colonies Rapa (ug/ml) Supplemental Figure 2. CFU plating efficiency and distribution of rapamycin-treated CB CD34 + cells. To determine the effect of rapamycin on CFU plating efficiency and progenitor cell type distribution, CB CD34 + cells were pre-stimulated and transduced in the presence of DMSO or rapamycin, using the transduction protocol presented in Figure 1A and Methods. Following transduction, cells were washed and seeded in methylcellulose medium for colony formation. Colonies were scored days post plating. (A) CFU plating efficiency, calculated as percentage of colony formation from the number of input cells, for CD34 + cells treated with DMSO only, 1, or 2 ug/ml of rapamycin. (B) CFU colony type distribution of (A), reflective of the distribution of progenitor cell types in input cells. GEMM = Granulocyte, Erythrocyte, Macrophage, Megakaryocyte, M = Macrophage, GM = Granulocyte, Macrophage, and BFU-E = Burst-Forming Unit-Erythroid. (C) Percentage of GEMM multipotent colonies out of total colonies from (B). Data are pooled from 3-5 independent experiments, each with different CB donors and done in triplicate. Lines represent group mean and error bars represent S.D. * denotes statistical significance of p<.5 from a parametric two- tailed unpaired Student s T test.

12 A CB CD34 + cells transduced with VSV-G LV MFI non-stimulated stimulated non-stimulated stimulated B BM CD34 + cells transduced with VSV-G LV C CB CD34 + cells transduced with LASV LV non-stimulated stimulated 1 2 Rapa (ug/ml) Supplemental Figure 3. Rapamycin-mediated transduction increase is augmented by cytokine stimulation, and is independent of cell source and LV pseudotype. (A) Human CB CD34 + cells were transduced with CG-UbiC-EGFP, MOI=5. Black triangles, DMSO only; red circles, 1 μg/ml rapamycin. (B) Human bone marrow (BM) CD34 + cells were transduced with CG-UbiC-EGFP, MOI=25. Black, DMSO only; red circles, 1 μg/ml rapamycin; orange squares, 2 μg/ml rapamycin. (C) Human CB CD34 + cells were transduced with Lassa virus envelope (LASV)-pseudotyped LV, MOI=3. Data are duplicate transductions from one experiment representative of at least two experiments with different CB donors. Lines represent group mean.

13 A Bone Marrow Spleen 1 1 % Donor derived Rapa B Total BM BM HSCs Lin - BM 8 p=.7 8 p= Viral copy number Rapa C B cells T cells Monocytes Neutrophils p=.11 1 * p= * Rapa Supplemental Figure 4. Increased EGFP marking in mouse hematopoietic lineages arising from rapamycin treated and LV-transduced Lin - cells in secondary recipient mice. 1x1 7 bone marrow (BM) cells from each primary recipient of LV transduced Lin - BM cells shown in Figure 2 were transplanted into lethally irradiated, congenically disparate recipient mice to assess the capacity for secondary engraftment and EGFP marking. Primary recipients were sacrificed at 11 weeks (grey triangles/open red circles) or 16 weeks (black triangles/closed red circles) post transplant. Secondary recipients were sacrificed at 1 or 45 weeks post transplant (for the 11 week or 16 week primary donors, respectively) and BM and spleen were analyzed for specified cell populations. (A) BM and splenic engraftment levels in secondary recipients, represented by the percentage of donor derived cells. (B) Left two panels, percentages of donor derived, EGFP expressing cells in total BM or Lin - Sca-1 + c-kit + HSCs. Right panel, LV copy number in Lin - BM cells. (C) EGFP expression in donor derived splenic B cells (B22 + ), T cells (B22 - CD4 + or CD8 + ), monocytes (B22 - CD11b + GR1 lo ), and neutrophils (B22 -

14 CD11b + GR1 + ). Each point represents one mouse. For all panels, lines represent group mean and error bars represent S.D. * denotes statistical significance of p<.5 from a parametric two- tailed unpaired Student s T test. p values are shown for results approaching statistical significance.

15 A Input CD34 + cells B Input CD34 + cells % of total colonies 1 5 CFU-GEMM CFU-M CFU-GM BFU-E Liquid culture CFU 1 2 Rapa (ug/ml) C Spleen 1 ** 1 p= MFI Rapa (ug/ml) 1 2 D Splenic myeloid cells Splenic B cells Thymic T cells 1 p=.57 p=.1 p=.71 1 ** 1 p= Rapa (ug/ml) E BM from secondary NSG mouse transplants N.E. N.E. : no engraftment 2 1' 2' 1' 2' - Rapa + Rapa

16 Supplemental Figure 5. Increased EGFP marking in input CD34 + cells used for establishing humanized NSG mice, in human myeloid and lymphoid cells obtained from spleens and thymi of primary recipient NSG mice, and in human cells obtained from the bone marrow of secondary recipient NSG mice. (A) EGFP marking in CD34 + cells used for establishing the humanized NSG mice in Figure 3. Human CB CD34 + cells were transduced with CG-UbiC-EGFP, MOI=25, in the presence of DMSO only, 1, or 2 μg/ml rapamycin, using the transduction protocol presented in Figure 1A. Aliquots of transduced cells were set aside and cultured in vitro in either liquid or CFU medium. For liquid culture, cells were cultured for 13 days and EGFP cell marking was determined by flow cytometry. For CFU assay, 1 cells per plate were seeded in triplicate plates in methylcellulose medium, and colonies were scored 15 days post plating for colony type and EGFP colony marking by microscopy. (B) Distribution of colony types from panel A. (C) EGFP marking and MFI in total spleen human CD45 + cells, from NSG mice discussed in Figure 3. (D) EGFP marking in splenic myeloid cells (hcd33 + ) and B cells (hcd19 + ), and thymic T cells (hcd3 + ), from NSG mice discussed in Figure 3. (E) Human CB CD34 + cells were transduced with CG-UbiC-EGFP, MOI=5, in the presence of DMSO or 1 μg/ml rapamycin. Cells were washed and injected into sublethally irradiated primary NSG recipients (1 ). After 12 weeks, BM cells from each mouse were isolated, equally divided, and injected into two sublethally irradiated secondary recipients (2 ). Mice were sacrificed 12 weeks post transplant and EGFP marking was determined in BM hcd45 + cells. N.E. = no engraftment. Each point represents one mouse. For all panels, lines represent group mean and error bars represent S.D. ** denotes statistical significance of p<.1 from a parametric two- tailed unpaired Student s T test. P-values are shown for results approaching statistical significance.

17 Supplemental Figure 6. LV cores do not co-localize with cytoplasmic LC3B+ foci. Human CB + CD34 cells were transduced with CG-UbiC-EGFP carrying GFP- Vpr protein for intracellular visualization, MOI=25, in the presence of PBS only (no drug), 1 µm chloroquine (CQ), 25 µm Tatbeclin 1, DMSO only (-Rapa), or 1 µg/ml rapamycin (+Rapa). After either 4 or 12 hours of transduction, as indicated above each panel, cells were fixed, permeabilized, and stained for LC3B.

18 LV core localization is tracked by fluorescence of the core-associated GFP-Vpr protein; representative LV cores are indicated with green arrows. Cell nuclei are visualized by staining with Hoechst and are outlined in white dashed circles. Presence of cytoplasmic punctate LC3B foci indicates autophagy induction; representative LC3B foci are indicated with red dashed arrows. Diffuse LC3B staining in the nuclei arise from nucleoplasmic localization of LC3 and does not indicate autophagy activation 17. Green, GFP-Vpr; red, LC3B; blue, Hoechst

19 A 6 B 1 C WT G89V MOI p21 D DNA RNA E 6h 12h 8 Percentages of CD34 + cells at stages of cell cycle at 12h EGFP marking of cells treated and transduced per diagram above 6 % of total cells G/G1 S G Rapa + Rapa -6h -4h -2h +2h +4h +6h Supplemental Figure 7. Rapamycin- mediated transduction enhancement is not due to LV capsid interaction, cellular p21 content, or changes in the cell cycle. (A) Human CB CD34 + cells were transduced with CG-UbiC-EGFP carrying wild type or G89V capsid protein, MOI=25, in the presence or absence of 1 μg/ml rapamycin or 1 nm cyclosporine A (CsA). Black triangle, -Rapa -CsA; black square, -Rapa +CsA; red circle, +Rapa -CsA; red square, +Rapa +CsA. (B) Human CB CD34 + cells were transduced with LV carrying wild type or PQVI capsid protein 18, in the presence or absence of 1 μg/ml rapamycin. Black triangle, WT -Rapa; black square, PQVI -Rapa; red circle, WT +Rapa; red square, PQVI +Rapa. (C) Human CB CD34 + cells were treated with rapamycin or DMSO for 12 hours, then stained for intracellular p21 protein and analyzed by flow cytometry. Black, DMSO; red, 1 μg/ml rapamycin. (D) (Upper panel): Human CB CD34 + cells were treated with rapamycin or DMSO for 6 or

20 12 hours, then stained with Hoechst for DNA content and Pyronin Y for RNA content and analyzed by flow cytometry. (Lower panel): Cell cycle distribution by DNA staining at 12 hours was quantified in FlowJo and plotted. Black, DMSO; red, 1 μg/ml rapamycin. Data are pooled from two independent experiments, each with different CB donors. (E) (Upper panel): Human CB CD34 + cells were transduced with CG-UbiC-EGFP, MOI=25, and treated with 1 μg/ml rapamycin at time intervals shown. Following pre- treatment with rapamycin, cells were washed before adding LV. Following transduction, cells were washed and cultured for 13 days before assessing EGFP expression. (Lower panel): EGFP expression of cells treated and transduced for the time intervals shown in the upper panel. X-axis indicates the time of rapamycin treatment initiation. -Rapa, -12h treatment with DMSO only; +Rapa, -12h treatment with rapamycin. Data are triplicate transductions from one experiment representative of three experiments with different CB donors. For all panels, lines represent group mean.

21 A hu CD34 + cells B hu CD34 + cells LV copies/cell MOI Early RT Late RT C mu Lin - cells D mu Lin - cells LV copies/cell Day 2 Day 8 Day 2 Day 8 Supplemental Figure 8. Rapamycin increases transduction of human CB CD34 + cells and mouse BM Lin - + cells with IDLV. (A) Human CB CD34 cells were transduced with IDLV-RRL-MND- GFP, MOI=5, 25, or 5, and EGFP cell marking was examined by flow cytometry at 3 and 9 days post transduction. Black, DMSO only; red, 1 μg/ml rapamycin; solid line, 3 days; dashed line, 9 days. (B) Human CB CD34 + cells were transduced with IDLV-RRL-MND-GFP, MOI=5, in presence of DMSO or 1 μg/ml rapamycin. Cells were harvested 12 hours post transduction, and copy numbers of HIV-1 early (strong stop DNA) and late (full-length DNA) reverse transcripts were determined by qpcr. Data are representative of three experiments with different CB donors. (C) Mouse Lin - cells were transduced with IDLV-RRL-MND-GFP, MOI=15. EGFP expression was examined by flow cytometry at 2 and 8 days post transduction, and (D) Copy numbers of total HIV-1 reverse transcripts were determined by qpcr. Black, DMSO only; red, 5 μg/ml rapamycin. Data shown are representative of two independent experiments.

22 A hu CD34 + cells B mu Lin - cells Baf (nm) 1 1 Baf (nm) Supplemental Figure 9. Rapamycin-mediated transduction enhancement targets ph-dependent endocytic entry and is blocked by bafilomycin A1 (Baf). (A) Human CB CD34 + cells were transduced with CG-UbiC-EGFP, MOI=25, in the presence or absence of rapamycin and Baf. Black, DMSO only; pink, 5 μg/ml rapamycin; red, 1 μg/ml rapamycin. Data are duplicate transductions from one experiment representative of three experiments with different CB donors. (B) Mouse BM Lin - cells were transduced with RRL-MND-GFP, MOI=2.5, in the presence or absence of rapamycin and Baf. White, DMSO only; pink, 1 μg/ml rapamycin; red, 5 μg/ml rapamycin. Data are pooled from two independent experiments, each with different donors and done in duplicate. For all panels, lines represent group mean and error bars represent S.D.