CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to USSN 62/462,293, filed on February 22, 2017, and to USSN 62/430,231, filed on December 5, 2016, both of which are incorporated herein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[ Not Applicable ]

BACKGROUND

[0002] Lentiviral vector (LV)-based gene-modification of autologous

hematopoietic stem cells (HSC) has proven to be a successful clinical strategy for treatment of genetic blood diseases (Boztug et al. (2010) N EnglJ Med. 363(20): 1918- 1927; Aiuti et al. (2009) N EnglJ Med. 360(5): 447-458; Aiuti et al. (2013) Science. 341(6148): 1233151; Candotti et al. (20\2) Blood. 120(18): 3635-3646; Hacein-Bey- Abina et al. (2010) N EnglJ Med. 363(4): 355-364; Kang et al. (2010) Blood,

115(4):783-791. doi: 10.1182/blood-2009-05-222760). In this approach, bone marrow (BM) or mobilized peripheral blood (mPB) is collected from a patient, enriched for HSC, transduced with LV encoding the correct genetic sequence, and transplanted back into the patient. After transplant, HSC engraft in the BM and produce mature, gene-corrected hematopoietic cells ideally throughout the patient's lifetime.

[0003] Current clinical HSC enrichment protocols use immunomagnetic beads

(IB) to select for cells expressing the surface marker CD34 prior to LV-transduction. This method may be inefficient in a gene therapy setting, as the majority of CD34 ⁺ cells are short-term progenitor cells with a limited post-transplant lifespan. Further isolation of HSC from short-term progenitors could improve LV economy by only transducing cells that can endure for the lifetime of the patient. As clinical grade vector production is expensive and often limited by commercial manufacturing capacity, reducing the amount of LV required per patient could improve the clinical and commercial feasibility of gene therapy. SUMMARY

[0004] We postulated that purifying CD34 ⁺ CD38 ^" cells would further enrich for

HSC, reduce the absolute number of cells to be transduced with LV, and reduce the total LV dose required per patient. Importantly, these benefits could be achieved while still retaining the target HSC required for long-term clinical benefit after transplantation.

[0005] While clinical transplant of CD34 ⁺ cells is common, little is known about the feasibility and efficacy of using more purified stem cell transplants (Tricot et al. (1988) Blood, 91(12): 4489-4495; Negrin et al. (2000) Biol. Blood Marrow Transpl. 6(3): 262-271; Michallet et al. (2000) Exp. Hematol. 28(7): 858-870). We have demonstrated efficient LV transduction of highly purified, Fluorescence Activated Cell Sorting (FACS)-sorted CD34 ⁺ CD38 ^" cells from cord blood (CB) (Baldwin et al. (2015) Stem Cells. 33(5): 1532-1542). Described herein is an IB-based method to purify CD34 ⁺ CD38 ^" cells from human BM and mPB samples with focus on practical implementation in a clinical setting. We demonstrate the capacity of this method to reduce LV dose by ~ 10-fold while maintaining early post-transplant recovery and long- term engraftment of gene-modified cells. These findings have the potential to reduce the total LV required per patient and improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.

[0006] Various embodiments cnetemplated herein may include, but need not be limited to, one or more of the following:

[0007] Embodinemtn 1 : A method of enriching a population of mammalian hematopoietic stem cells for CD34+/CD38- stem cells, said method comprising:

[0008] contacting a mammalian hematopoietic tissue sample with a first antibody that binds to CD38 and capturing and removing cells in said sample bound by said first antibody to produce a first cell population that is partially or fully depleted of CD38+ cells; and

[0009] contacting said first cell population with a second antibody that binds to CD34 and isolating cells bound by said second antibody to produce a second population of cells that is enriched for CD34 ⁺ CD38 ^" stem cells. [0010] Embodiment 2: The method of embodiment 1, wherein said

hematopoietic tissue sample and/or said first cell population is contacted with a third antibody that binds to a granulocyte marker and capturing and removing cells bound by said third antibody to produce said first cell population wherein said first cell population is also partially or fully depleted of granulocytes.

[0011] Embodiment 3 : The method of embodiment 2, wherein said

hematopoietic tissue sample is simultaneously contacted with said first antibody and third second antibody.

[0012] Embodiment 4: The method of embodiment 2, wherein said

hematopoietic tissue sample is sequentially contacted with said first antibody and said third antibody.

[0013] Embodiment 5: The method of embodiment 2, wherein said first cell population is contacted with said third second antibody.

[0014] Embodiment 6: The method according to any one of embodiments 2-5, wherein said third antibody is an antibody that binds a granulocyte marker selected from the group consisting of CD15, CD16, CDl lb, CD33, and CD66.

[0015] Embodiment 7: The method of embodiment 6, wherein said third antibody is an antibody that binds to CD 15.

[0016] Embodiment 8: The method according to any one of embodiments 1-7, wherein said first antibody is attached to a magnetic bead, and said capturing and removing cells in said sample bound by said first antibody comprises capturing said magnetic bead using a magnetic field. [0017] Embodiment 9: The method according to any one of embodiments 1-7, wherein said first antibody is attached to a label, and said label is bound by a label- specific antibody attached to a magnetic bead, and said capturing and removing cells in said sample bound by said first antibody comprises capturing said magnetic bead using a magnetic field. [0018] Embodiment 10: The method of embodiment 9, wherein said label comprises phycoerythrin (PE), and said label-specific antibody is an anti-PE antibody.

[0019] Embodiment 11 : The method according to any one of embodiments 9-10, wherein said sample comprises mobilized peripheral blood and the label-specific antibody bead concentration is about 1 : 10. [0020] Embodiment 12: The method according to any one of embodiments 9-10, wherein said sample comprises bone marrow and the label-specific antibody bead concentration is about is about 1 :25.

[0021] Embodiment 13 : The method according to any one of embodiments 8-12, wherein said magnetic field is produced by a magnet.

[0022] Embodiment 14: The method according to any one of embodiments 8-12, wherein said magnetic field is produced by a magnetic column.

[0023] Embodiment 15: The method according to any one of embodiments 1-7, wherein said first antibody is attached to an affinity column and said capturing and removing cells in said sample bound by said first antibody comprises passing said sample over or through said affinity column.

[0024] Embodiment 16: The method according to any one of embodiments 1-7, wherein said first antibody is attached to an avidin and said capturing and removing cells in said sample bound by said first antibody comprises passing the antibody bound cells over a substrate comprising biotin.

[0025] Embodiment 17: The method according to any one of embodiments 1-7, wherein said first antibody is attached to a biotin and said capturing and removing cells in said sample bound by said first antibody comprises passing the antibody bound cells over a substrate comprising an avidin. [0026] Embodiment 18: The method according to any one of embodiments 2-17, wherein said third antibody is attached to a magnetic bead, and said capturing and removing cells in said sample bound by said third antibody comprises capturing said magnetic bead using a magnetic field to remove said granulocytes.

[0027] Embodiment 19: The method of embodiment 18, wherein said magnetic field is produced by a magnet.

[0028] Embodiment 20: The method of embodiment 18, wherein said magnetic field is produced by a magnetic column.

[0029] Embodiment 21 : The method according to any one of embodiments 2-17, wherein said third antibody is attached an affinity column and said capturing and removing cells in said sample bound by said third antibody comprises passing said sample over or through said affinity column to remove said granulocytes. [0030] Embodiment 22: The method according to any one of embodiments 2-17, wherein said third antibody is attached to an avidin and said capturing and removing cells in said sample bound by said third antibody comprises passing the antibody bound cells over a substrate comprising biotin to remove said granulocytes. [0031] Embodiment 23 : The method according to any one of embodiments 2-17, wherein said third antibody is attached to a biotin and said capturing and removing cells in said sample bound by said third antibody comprises passing the antibody bound cells over a substrate comprising an avidin to remove said granulocytes.

[0032] Embodiment 24: The method according to any one of embodiments 1-23, wherein said second antibody is attached to a magnetic bead, and said isolating cells bound by said second antibody comprises capturing said magnetic bead using a magnetic field to produce said second population of cells that is enriched for CD34 ⁺ CD38 ^" stem cells.

[0033] Embodiment 25: The method according to any one of embodiments 1-23, wherein said second antibody is attached to a label, and said label is bound by a label- specific antibody attached to a magnetic bead, and said isolating cells bound by said second antibody comprises capturing said magnetic bead using a magnetic field to produce said second population of cells that is enriched for CD34 ⁺ CD38 ^" stem cells.

[0034] Embodiment 26: The method according to any one of embodiments 24- 25, wherein said magnetic field is produced by a magnet.

[0035] Embodiment 27: The method according to any one of embodiments 24-

25, wherein said magnetic field is produced by a magnetic column.

[0036] Embodiment 28: The method according to any one of embodiments 1-23, wherein said second antibody is attached to an affinity column and said isolating cells bound by said second antibody comprises passing said cells over or through said affinity column and eluting the captured cells from said column to produce said second population of cells that is enriched for CD34 ⁺ CD38 ^" stem cells.

[0037] Embodiment 29: The method according to any one of embodiments 1-28, wherein said hematopoietic tissue comprises a tissue selected from the group consisting of cord blood, bone marrow, and mobilized peripheral blood.

[0038] Embodiment 30: The method of embodiment 29, wherein said hematopoietic tissue comprises mobilized peripheral blood. [0039] Embodiment 31 : The method of embodiment 29, wherein said hematopoietic tissue comprises bone marrow.

[0040] Embodiment 32: The method according to any one of embodiments 1-31, wherein said mammalian tissue sample comprises a human tissue sample. [0041] Embodiment 33 : The method according to any one of embodiments 1-31, wherein said mammalian tissue sample comprises a non-human mammalian tissue sample.

[0042] Embodiment 34: The method according to any one of embodiments 1-33, wherein said method is performed to provide less than about 30-fold reduction in total cell number, or less than about 20-fold reduction in total cell number, or less than about 15-fold reduction in total cell number, or about a 10-fold reduction in total cell number.

[0043] Embodiment 35 : A population of stem cells enriched for CD34+/CD38- stem cells.

[0044] Embodiment 36: The population of stem cells, wherein said population is produced by a method according to any one of embodiments 1-34.

[0045] Embodiment 37: The population according to any one of embodiments

35-36, wherein said population of cells comprises CD34 ⁺ cells is within the lowest 10% or within the lowest 6% of CD38 negativity in a population of bone marrow cells.

[0046] Embodiment 38: The population according to any one of embodiments 35-37, wherein said cells show delayed myeloid reconstitution that can be rescued by the addition of non-transduced CD38 ⁺ cells.

[0047] Embodiment 39: The population according to any one of embodiments

35-38, wherein lentiviral vector (LV) transduction of said CD34 ⁺ CD38 ^" cells coupled with co-transplantation with non-transduced CD38 ⁺ cells provides long-term gene- marked engraftment comparable to modification of bulk CD34 ⁺ cells, while utilizing significantly less LV.

[0048] Embodiment 40: The population of embodiment 39, wherein said transduction is accomplished while utilizing at least 5-fold, or about 7-fold less LV.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 illustrates results of an enrichment methods that includes the addition of a granulocyte depletion bead (e.g., CD 15). [0050] Figure 2 schematically illustrates one embodiment of enrichment methods described herein.

[0051] Figures 3A-3B show long-term repopulating activity of CD34 ⁺ CD38 subsets. Fig. 3 A) Experimental set up. 1. FACS sort: BM cells were magnetically enriched for CD34 and sorted by FACS into distinct populations of increasing CD38 expression. Each gate contains an equivalent number of cells. 2. LV transduction: Cells from each interval were transduced with one of three LV expressing the fluorescent proteins mCitrine, mStrawberry, or mCerulean. 3. Transplant: A combination of cells from each sorted interval (each differentially labeled) were combined and competitively transplanted into sub-lethally irradiated NSG mice. Each mouse received an equal number of cells from each sorted fraction. 4. BM analysis: NSG BM was analyzed at 16-18 weeks for the presence of fluorescently labeled hCD45 ⁺ cells. Fig. 3B) Relative abundance of labeled cells in NSG BM. Two separate transplants were performed using different intervals of CD38 expression (top and bottom). Initial sort for CD38 expression is shown in the left panels. At 16-18 weeks post-transplant, fluorescent, LV- labeled hCD45+, hCD19+, and hCD33+ cells were analyzed, y axis represents the relative frequency of each sorted and/or labeled CD38 fraction as a percent of all fluorescently labeled cells. Bars represent mean ± SEM. n = 7 mice for transplant 1; n = 3 mice for transplant 2. [0052] Figures 4A-4B show that titration of CD38 immunomagnetic labeling influences recovery and enrichment of CD34 ⁺ CD38 ^" cells. Fig. 4A) CD38 depletion of BM mononuclear cells (MNCs) using 3 different concentrations of anti-PE beads. Plots are gated to show CD34 ⁺ cells present in starting MNCs (far left), and after column separation into CD38 ⁺ (top) and CD38 ^" (bottom) fractions. Gates show CD34 ⁺ CD38 ^" cells (defined as the lowest 6% of CD38 negativity) and list absolute counts of

CD34 ⁺ CD38 ^" cells in each population. Fig. 4B) Efficiency of CD38 depletion step at different PE bead concentrations. Bar graphs show "Percent recovery" and "Fold- enrichment" of CD34 ⁺ CD38 ^" cells before and after CD38 depletion using 3 different anti-PE bead concentrations. Bars represent mean ± SEM, data represent 3 independent experiments for each CB, BM, and mPB.

[0053] Figures 5A-5C show that co-depletion of CD15 ⁺ and CD38 ⁺ cells leads to greater CD34 ⁺ CD38 ^" purity. Fig. 5 A) CD34 ⁺ CD38 ^" cells were purified from CB MNCs using one of two methods: CD38 depletion followed by CD34 selection (top) or CD38/CD15 co-depletion followed by CD34 selection (bottom). Left-most FACS plot shows CD34 and CD38 staining of all viable leukocytes (CD45 ⁺ DAPI ^" ) isolated by either method. Histograms indicate the percentage of CD15 ⁺ and CD34 ⁺ cells within total viable leukocytes isolated by each method. Fig. 5B) IB purification of BM#1. Left panel shows CD34 purification, and right panel shows parallel CD34 ⁺ CD38 ^" purification (using CD38 depletion only). FACS plots show viable leukocytes (CD45 ⁺ DAPF) isolated by each method. Gates show CD34 ⁺ CD38 ^" cells (top) and granulocytes (defined by high SSC, bottom) purified by each method. Absolute cell counts are listed in parentheses. Fig. 5C) IB purification of BM#2. Left panel shows CD34 purification, and right panel shows parallel CD34 ⁺ CD38 ^" purification (using CD38/CD15 co-depletion). FACS plots show viable leukocytes (CD45 ⁺ DAPF) isolated by each method. Gates show CD34 ⁺ CD38 ^" cells (top) and granulocytes (defined by high SSC, bottom) purified by each method. Absolute cell counts are listed in parentheses.

[0054] Figures 6A-6D show a comparison of CD34 ⁺ single-step IB purification vs CD34 ⁺ CD38 ^" dual IB purification. Fig. 6A) Comparison of a single bone marrow sample purified in parallel by either CD34+ single step IB purification (top) or

CD34 ⁺ CD38 ^" dual IB purification (bottom). Gates show CD34 ⁺ CD38 ^" cells (defined as the lowest 6% of CD38 negativity) with absolute cell counts are listed parentheses. Fig. 6B) Recovery and enrichment of CD34 ⁺ CD38 ^" cells obtained from IB purification of BM and mPB MNCs. 5 BM samples and 3 mPB sample from independent donors were purified in parallel by either CD34 ⁺ single-step IB purification or CD34 ⁺ CD38 ^" dual IB purification. For each sample, CD34 ⁺ CD38 ^" cells were defined as those with the lowest 6% of CD38 negativity within CD34 ⁺ cells. Recovery indicates the percentage of CD34 ⁺ CD38 ^" cells recovered after purification from starting MNCs, and is calculated for both CD34 ⁺ single-step (row 1) and CD34 ⁺ CD38 ^" dual IB purification (row 2). Fold- reduction (row 3) indicates the reduction in the total number of cells purified by

CD34 ⁺ CD38 ^" dual IB selection as compared to standard CD34 selection. This represents the clinically relevant reduction in the number of cells to be transduced and concordant vector dose reduction. Fold-enrichment (row 4) represents the enrichment of

CD34 ⁺ CD38 ^" cells as a percentage of total cells obtained by CD34 ⁺ CD38 ^" dual IB purification compared to standard CD34 ⁺ IB purification (see Table 1 for absolute cell counts for each IB purified fraction). Fig. 6C) Poisson statistical analysis of limiting dilution NSG xenotransplant assay of IB purified BM. Plots demonstrate SRC frequency of CCL-UBC-mCitrine transduced CD34 ⁺ or CD34 ⁺ CD38 ^" IB purified cells (n = 5-7 mice transplanted at each cell dose per condition, see Figure 12 for individual values). Y axis shows the percentage of recipient NSG mice containing <0.015% hCD45 ⁺ cells (non-engrafted) in the BM at 16 weeks post-transplantation versus the number of cells injected per mouse (X axis). Fig. 6D) In vivo VCN analysis. NSG mice were transplanted with IB-purified CD34 ⁺ or CD34 ⁺ CD38 ^" cells transduced with CCL-UBC- mCitrine LV. 16 weeks post-transplant, engrafted human cells in NSG BM were analyzed for VCN by ddPCR. Data represent 3 independent experiments performed using bone marrow from independent donors, n= 21-23 mice/group, n.s. not significant.

[0055] Figures 7A-7C show early myeloid potential of IB purified CD34 ⁺ and CD34 ⁺ CD38 ^" cells. Fig. 7A) Early myeloid engraftment of CD34 ⁺ and CD34 ⁺ CD38 ^" IB purified cells. Peripheral blood hCD45 ⁺ hCD33 ⁺ cells/mL were quantified at 4 weeks from NSG-SGM3 mice transplanted with CD34 ⁺ cells or increasing doses of

CD34 ⁺ CD38 ^" cells IB purified from BM. Cell doses for each group reflect the total cells obtained from IB purification from a standard volume of marrow (marrow equivalent or ME), n = 3 mice/group. Fig. 7B) CD38 add-back experimental set-up. BM MNCs were IB-depleted of CD3 ⁺ T cells to avoid xenogeneic graft versus host disease. CD3 ^" cells were further purified to obtain CD34 ⁺ , CD34 ⁺ CD38 ^" , or CD38 ⁺ populations. CD34 ⁺ and CD34 ⁺ CD38 ^" cells were transduced with a CCL-UBC-mCitrine LV, while CD38 ⁺ cells were cultured in parallel without the addition of LV. 4 experimental arms were transplanted, with each mouse receiving 1 marrow equivalent of the pictured graft. Gray boxes represent UBC-mCitrine transduced populations while unfilled boxes represent non-transduced populations. Fig. 7C) Early myeloid engraftment of IB purified cells in NSG-SGM3 mice. Two transplants were performed using the 4 arms described in Fig. 7B. Flow plots show CD34 ⁺ or CD34 ⁺ CD38 ^" IB-purified cells prior to transplant. Bar graphs represent mean ± SEM of myeloid engraftment in peripheral blood. Gray bars represent total myeloid engraftment and overlay of black bars represent gene-marked (mCitrine+) myeloid engraftment. Myeloid engraftment is expressed as the percent of hCD45 ⁺ CD33 ⁺ /CD66b ⁺ /CD15 ⁺ cells out of total CD45 ⁺ (human and murine) cells. Each graph represents 1 experiment performed using bone marrow from independent donors with n=4-5 mice/arm. n.s. not significant, *P < .05, **P < .01.

[0056] Figure 8A-8C show long-term engraftment of IB purified cells. Fig. 8A)

Long-term engraftment of IB purified cells in NSG BM (top) or spleen (bottom) 16 weeks post-transplant. Grey bars represent mean ± SEM of total human engraftment (%hCD45 ⁺ ) while black bars represent gene-marked engraftment (%hCD45 ⁺ mCitrine ⁺ ). Engraftment is expressed as the percent of hCD45 ⁺ or hCD45 ⁺ mCitrine+ cells out of total CD45 ⁺ (human and murine) cells. Data represent 2 experiments using bone marrow from independent donors with a total of 8-9 mice/arm. n.s. not significant, *P < .05, ***P<0.001. Fig. 8B) Lineage analysis of total hCD45 ⁺ in NSG bone marrow 16 weeks post-transplant. Bars show mean ± SEM of each lineage (CD19 ⁺ B cells, CD33 ⁺ myeloid cells, CD3 ⁺ T cells, and CD34 ⁺ HSPC) expressed a percent of total hCD45 ⁺ cells. Data represent 2 experiments using bone marrow from independent donors with a total of 8-9 mice/arm. n.s. not significant, *P < .05. Fig. 8C) PB engraftment over time after transplant of UBC-mCitrine transduced 34 ⁺ cells (filled circles) or UBC-mCitrine transduced CD34 ⁺ CD38 ^" with add-back of non-transduced CD38 ⁺ cells (open squares). Left panel shows total PB engraftment (%hCD45 ⁺ ) while right panel shows gene-marked engraftment (%hCD45 ⁺ mCitrine ⁺ ). n.s. not significant, *P < .05.

[0057] Figure 9 illustrates a proposed clinical cell processing pathway. BM or mPB will be RBC/platelet depleted, followed by labeling with CD38-PE, anti-PE beads, and anti-CD 15 beads. The CD38 ⁺ /CD 15 ⁺ fraction (bound to the column) will undergo short-term culture followed by cryopreservation and release testing. The CD387CD15 ^" fraction (column flow-through) will be labeled with anti-CD34 beads, followed by selection of CD34 ⁺ CD38 ^" cells. CD34 ⁺ CD38 ^" cells will undergo transduction with lentiviral vector followed by cryopreservation and release testing. After successful release testing, transduced CD34 CD38 ^" cells and non- transduced CD387CD15 ⁺ cells will be thawed and co-transplanted into a conditioned patient.

[0058] Figure 10A shows flow cytometry gating for BM CD34 ⁺ cells prior to defining CD38 intervals. Far right panel shows CD34 ⁺ cells used to determine intervals of CD38 expression (e.g., CD34 ⁺ CD38° ^"3% ). Figure 10B FACS plot shows sorted CD38 intervals relative to the max fluorescence of the CD38 FMO control (red line). Figure IOC bar graphs show transduction efficiency for each vector (mStrawberry, mCitrine, or mCerulean). Transduced cells were cultured for 14 days and transduction efficiency was measured by flow cytometry. Figure 10D graphs show long-term (16-18 week) fluorescent marking of hCD45 ⁺ cells in BM by individual mouse. Each bar represents a single mouse and colors within each bar represent the relative contribution of each labeled fraction. Color coding represents the LV used for each fraction (green=mCitrine, blue=mCerulean, red=m Strawberry). Lower chart shows the color assigned to each sorted fraction transplanted. Colors were rotated among fractions and mice to account for differences in transduction efficiency among the vectors. Data show that

repopulating activity does not appear to be influenced by color choice

[0059] Figure 11 shows gating for enumeration of CD34+CD38- in MACS purified fractions. CD34 ⁺ CD38 ^" backgating is shown in red. [0060] Figure 12A and 12B shows limiting Dilution Analysis of CD34+ and

CD34+CD38- IB purified cells. Fig. 12A) Engraftment (%hCD45 ⁺ ) for each mouse at 3 different cell doses. Each cell close represents 34 ⁺ or 34 ⁺ 38 ^" cells purified from an equivalent volume of marrow. Dashed line depicts the engraftment cut-off of 0.015% hCD45 , which was arbitrarily chosen as -20 times the mean engraftment measured in 5 non-transplanted controls. Tables show cell numbers for each transplanted group and the number of mice with engraftment >0.015% at each cell dose. Fig. 12B) FACS plots show detection of hCD45+ cells at levels near cut-off for engraftment (0.015% hCD45 ⁺ ). A non-transplanted control is shown for comparison.

[0061] Figure 13 A shows the determination of human myeloid engraftment in peripheral blood by flow cytometry. Human myeloid engraftment is expressed as the percent of hCD45 ⁺ CD33/CD66b/CD15 ⁺ cells out of total CD45 ⁺ (human and murine) cells. Figure 13B shows CD38 addback into NSG mice. Gray bars represent mean i SEM of total human myeloid engraftment in peripheral blood

(%hCD45 ⁺ CD33/CD66b/CD15 ⁺ out of total CD45 ⁺ ). Overlay of black bars represent mCitrine ⁺ human myeloid cells (%mCitrine ⁺ hCD45"CD33/CD66b/CD 15 ⁺ out of total CD45 ⁺ ). Data represents 2 experiments using bone marrow from independent donors with n=9-10 mice/arm. To account for engraftment variability between donors, data from each experiment was normalized to mean CD34+ values for each experiment.

[0062] Figure 14 shows cell numbers for in vivo xenografts. DETAILED DESCRIPTION

[0063] Development of a method that further isolates HSC from short-term progenitors could improve efficiency by only transducing cells that will endure for the lifetime of the patient. CD38 is a cell surface marker expressed on late progenitors and mature cells with low/absent expression on HSC8. We postulated that purifying

CD34+CD38- cells would further enrich for HSC, reduce the absolute number of cells to be transduced with vector, and reduce the total vector dose required per patient. Importantly, these benefits could be achieved while still retaining the key target HSCs needed for a long-term clinical benefit after transplantation.

[0064] While CD34 selection of HSPC has been used for many years in a clinical transplant setting, little is known about the safety and feasibility of using more purified stem cell transplants. The majority of studies characterizing CD34+CD38- cells and their repopulation kinetics have been performed using cord blood. While this is a widely available and affordable source of stem cells for research use, the majority of clinical transplants are performed using adult sources of stem cells such as bone marrow or mobilized peripheral blood. [0065] Our lab has previously demonstrated that highly purified, FACS sorted

CD34+CD38- cells from cord blood can be transduced with lentiviral vector with high efficiency. Here, we demonstrate the pre-clinical development of an IB-based method to purify human CD34+CD38- cells from bone marrow and evaluate the capacity of our method to reduce vector dose in a clinically relevant model of BMT. Our findings have the potential to reduce total vector production required per patient and improve the clinical and commercial viability of gene therapy for SCD.

[0066] Here, we describe a strategy improve enrichment of HSC prior to genetic correction. To enrich for HSC, we have developed an immunomagnetic bead (IB) based, clinically applicable method to purify CD34 ⁺ 38 ^" cells from whole, human hematopoietic tissue (e.g,. cord blood, bone marrow, or mobilized peripheral blood).

[0067] In certain embodiments CD34+38- cells are purified using the following method: Whole hematopoietic tissue or mononuclear cells (MNC) are labeled with magnetic beads recognizing CD38 and magnetic beads recognizing any granulocyte marker (e.g., CD15, CD16, CDl lb, CD33, CD66). The CD38/granulocyte magnetically labeled cells are then applied to a magnetic column and the unlabeled fraction is collected. This unlabeled fraction has been depleted of CD38+ cells and granulocytes. The CD38/granulocyte depleted cells are then incubated with CD34 beads and applied to a magnetic column to select CD34 ⁺ cells. The resulting product is a highly purified population of CD34 ⁺ 38 ^" cells. [0068] Our method minimizes total cell processing time by depleting CD38 cells prior to CD34 selection. This avoids additional processing time and reagents required for magnetic bead removal. The alternative strategy of first selecting with CD34 beads would require magnetic beads to be removed from CD34+ cells prior to CD38 depletion. [0069] The addition of a granulocyte depletion bead (e.g., CD 15) is extremely useful to obtain a highly pure population of CD34+38- cells (see, e.g., Figure 1).

CD34 ⁺ 38 ^" cells are relatively rare (-0.1%) within CD38 depleted cells and greatly outnumbered by granulocytes. During the CD34 selection step, a portion of CD38- granulocytes non-specifically bind to CD34 magnetic beads or the magnetic column, and are co-purified along with CD34 ⁺ 38 ^" cells. This results in a final product containing both CD34+38- cells and a large number of contaminating granulocytes. Addition of a granulocyte depleting bead (e.g., CD 15) in the first depletion step removes

contaminating granulocytes and results in a highly purified population of CD34 ⁺ CD38 ^" cells.

[0070] CD34 ⁺ CD38 ^" cells have previously been shown to be highly enriched for long-term HSC, but isolation of this population has not previously been used in a clinical transplant setting. Current clinical protocols for gene therapy enrich HSCs from bone marrow using IB purification for CD34 ⁺ cells. However, the majority of CD34 ⁺ cells are committed progenitor cells that have a limited life-span (months) after transplant into a patient. Thus, enriching CD34 ⁺ cells prior to genetic correction is inefficient, as the majority of corrected cells will only provide short-term engraftment.

[0071] The methods described herein aims to reduce the total cell number to undergo gene correction and are believed to have many clinical advantages. If cells are genetically modified using viral vectors, a smaller number of absolute target cells to be transduced would require a lower vector dose and greatly reduce the high cost and labor demands associated with clinical grade vector production. If cells are genetically modified using electroporation of targeted nucleases (e.g., ZFN or CRISPR/Cas9), a smaller cell number could increase electroporation efficiency by decreasing cell processing scale and preserve cell viability by reducing total processing time.

Importantly, these clinical benefits could be achieved while still retaining the long-term HSC required for clinical efficacy after transplant.

[0072] While Fluorescence Activated Cell Sorting (FACS) has been used in a research setting to obtain highly purified CD34 ⁺ 38 ^" populations, it is believed the methods described herein are advantageous for the following, and other, reasons:

[0073] 1. Preliminary data suggests that recovery of IB -separated

CD34+38- cells is ~5 fold higher than FACS-separated cells; [0074] 2. IB-separation can quickly purify the large number of cells obtained from a clinical-scale harvest while FACS may require many hours to sort a large number of cells; the shorter processing time may better preserve stem cell capacity and improve clinical efficiency; [0075] 3. Clinical IB-based cell sorting platforms (e.g., Miltenyi' s

Prodigy) are currently FDA approved, while no FDA-approved clinical FACS sorter is currently available.

[0076] The described methods of purifying CD34 ⁺ 38 ^" cells can be used in any clinical application of gene therapy for genetic blood diseases. These methods may also be useful in a research setting, as they permit a user to obtain highly purified CD34 ⁺ 38 ^" populations from valuable clinical samples with greater efficiency and recovery than FACS.

[0077] While the methods described above are described with respect to the use of antibodies bound to magnetic beads, one of skill will recognize that a number of diferent "capture" methods can be utilized. For example in certain embodimetns the magnetic beads are attached to an antibody that binds CD38 and/or to an antibody that binds CD34, and, when utilized, to an antibody that binds to a granulocyte marker.

However in various embodiments, the antibody that binds CD38 and/or the antibody that binds CD34 can be attached to a label which is then bound by an antibody specific for that label (a label-specific antibody) attached to a magnetic bead. Any of a number of labels that can be specifically bound by an antibody are suitable. In certain embodiments the label is a fluorochrome (e.g., fluorescein isothiocyanate (FITC), R-phycoerythrin (R- PE), peridinin chlorphyll protein (Percp), allophycocyanin (APC), CY5, PE-Texas red, PE-CY5, PE-CY7, PerCP-CY5.5, APC-CY7, Alexa Fluor 350, 488, 633, 647, 680, 430, cascade yellow, cascade blue, and the like) and the magnetic bead is attached to an antibody that specifically binds to the fluorochrome. For example, in certain

embodiments, the antibody that binds (e.g., specifically binds) to CD38 is an anti-CD38- PE antibody that is bound by an anti-PE antibody attached to a magnetic bead.

[0078] In certain embodiments magnetic beads need not be used. For example in certain embodiments, the anti-CD38 antibody can be provided on an affinity column and passing the sample over the affinity column will retain cells displaying the CD38 marker and the material recovered will be partially or fully depleted for CD38+ cells. Similarly, in certain embodiments, an anti -granulocyte antibody can be provided on the same affinity column or on a second affinity column and passing the sample over the affinity column containing anti -granulocyte antibodies (e.g., anti-CD 15) provides a cell population that is additionally partially or fully depleted of granulocytes. In certain embodiments the second (anti-CD34) antibody can be provided on an affinity column. The CD38-depleted (and optionally granulocyte-depleted) sample can be passed over this affinity column which captures CD34+ cells. The captured cells can be eluted from the column to provide the enriched CD38 ^" CD34+ cell population.

[0079] In certain embodiments the anti-CD38 antibody and/or anti-CD34 antibody and/or anti-granulocyte marker antibody can be functionalized with an avidin or a biotin which is then captured by a substrate bearing the corresponding avidin or biotin. In certain embodiments the anti-CD38 antibody and/or the anti-CD34 antibody and/or the anti-granulocyte antibody is attached to a label that is then bound by a label-specific antibody (e.g., as described above) where the label specific antibody is attached to a biotin or to an avidin that can be captured by a substrate bearing the corresponding avidin or biotin.

[0080] Thus, in certain embodiments, methods are provided for enriching a population of mammalian hematopoietic stem cells for CD34 ⁺ /CD38 ^" stem cells, where the methods involve contacting a mammalian hematopoietic tissue sample with a first antibody that binds to CD38 (anti-CD38 Ab) and capturing and removing cells in the sample bound by the first antibody to produce a first cell population that is partially or fully depleted of CD38 ⁺ cells; and contacting the first cell population with a second antibody that binds to CD34 (anti-CD34 Ab) and isolating cells bound by the second antibody to produce a second population of cells that is enriched for CD34 ⁺ CD38 ^" stem cells. In certain embodiments the hematopoietic tissue sample and/or the first cell population is contacted with a third antibody that binds to a granulocyte marker (e.g. , anti-CD 15 Ab) and cells bound by the third antibody are captured and removed to produce the first cell population wherein the first cell population is also partially or fully depleted of granulocytes. In certain embodiments the tissue sample is simultaneously contacted with the anti-CD38 antibody and the anti-granulocyte marker antibody (e.g., anti-CD15 Ab). In certain embodiments the tissue sample is sequentially contacted with the anti-CD38 antibody and the anti-granulocyte marker antibody (e.g., anti-CD 15, anti- CD16, anti-CDl 1, anti-CD33, anti-CD66 antibodies, and the like). In certain

embodiments the tissue sample is first partially or fully depleted of granulocytes, while in other embodiments, the the tissue sample is first partially or fully depleted of CD38+ cells.

[0081] As noted above, in certain embodiments, the first antibody (anti-CD38

Ab) is attached to a magnetic bead, and capturing and removing cells in the sample bound by the first antibody comprises capturing said magnetic bead using a magnetic field. In certain embodiments the first antibody is attached to a label, and the label is bound by a label-specific antibody (e.g., an anti-PE Ab) attached to a magnetic bead, and the capturing and removing cells in the sample bound by the first antibody comprises capturing the magnetic bead using a magnetic field (e.g., produced by a magnetic column, an electromagnet, a permanent magnet, etc.). In certain embodiments the first antibody (anti-CD38 Ab) is attached to an affinity column and and capturing and removing cells in the sample bound by the first antibody comprises passing the sample over (the affinity matrix) or through the affinity column to remove cells in the sample bound captured by the affinity column. [0082] In certain embodiments the second antibody (anti-CD34 Ab) is attached to a magnetic bead, and isolating cells bound by the second antibody comprises capturing the magnetic bead using a magnetic field (e.g., produced by a magnetic column, an electromagnet, a permanent magnet, etc.) to produce the second population of cells that is enriched for CD34 ⁺ CD38 ^" stem cells. In certain embodiments the second antibody (anti-CD34 Ab) is attached to a label, and the label is bound by a label-specific antibody attached to a magnetic bead, and isolating cells bound by the second antibody comprises capturing the magnetic bead using a magnetic field (e.g., produced by a magnetic column, an electromagnet, a permanent magnet, etc.) to produce the second population of cells that is enriched for CD34 ⁺ CD38 ^" stem cells. In certain embodiments the second antibody (anti-CD34 Ab) is attached to an affinity column and isolating cells bound by the second antibody comprises passing the cells over or through the affinity column and eluting the captured cells from the column to produce the second population of cells that is enriched for CD34 ⁺ CD38 ^" stem cells.

[0083] In certain embodiments the third antibody (anti -granulocyte marker Ab) is attached to a magnetic bead, and and capturing and removing cells (e.g., granulocytes) in the sample bound by the second antibody comprises capturing the magnetic bead using a magnetic field. In certain embodiments the third antibody is attached to a label, and the label is bound by a label-specific antibody (e.g., an anti-PE Ab) attached to a magnetic bead, and the capturing and removing cells in the sample bound by the third antibody comprises capturing the magnetic bead using a magnetic field (e.g., produced by a magnetic column, an electromagnet, a permanent magnet, etc.). In certain embodiments the third antibody (anti-CD 15 Ab) is attached to an affinity column and capturing and removing cells in the sample bound by the third antibody comprises passing the sample over (the affinity matrix) or through the affinity column to remove cells in the sample bound captured by the affinity column.

[0084] In various embodiments the magnetic bead can be replaced by an avidin or a biotin in which case, capture is accomplished by contacting the immunocomplex with a substrate bearing the corresponding biotin or avidin.

[0085] It will be recognized that the methods described herein can be performed using tissue obtained from humans or from non-human mammals. Accordingly in certain embodiments, veterinary applications are contemplated. In certain embodiments the sample comprises a tissue selected from the group consisting of cord blood, bone marrow, and mobilized peripheral blood. In certain embodiments the stem cells contemplated herein expressly exclude human embryonic stem cells.

[0086] In certain embodiments popoulations of stem cells enriched for

CD34+/CD38- stem cells are contemplated. In various embodiments the population is produced by an enrichment method described herein. In certain embodiments the population of cells comprises CD34 ⁺ cells within the lowest 10% or within the lowest 6% of CD38 negativity in a population of bone marrow cells. In certain embodiments the cells show delayed myeloid reconstitution that can be rescued by the addition of non- transduced CD38 ⁺ cells. In certain embodiments lentiviral vector (LV) transduction of the CD34 ⁺ CD38 ^" cells coupled with co-transplantation with non-transduced CD38 ⁺ cells provides long-term gene-marked engraftment comparable to modification of bulk CD34 ⁺ cells, while utilizing significantly less LV.

[0087] The foregoing embodiments are illustative and non-limmiting. Using the teaching provided herein, numerous variations on the enrichment methods described herein will be available to one of ordinary skill in the art.

EXAMPLES

[0088] The following examples are offered to illustrate, but not to limit the claimed invention. Example 1

Enrichment of Hematopoietic Stem Cells Using Immunomagnetic Beads to

Facilitate Gene Therapy

[0089] Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising clinical strategy for the treatment of genetic diseases. Clinical trials are currently underway to treat hemoglobinopathies using a lentivirus expressing anti-sickling β ϋ or γ-globin genes. However, clinical scale production of globin vectors has proven difficult due to the large size and complexity of the human β-globin gene expression cassette. Vector products often have low titers and require large manufacturing volumes to treat a single patient. We hypothesized that less vector per patient could be used by further purifying hematopoietic stem cells (HSC) beyond standard CD34+ selection prior to gene modification and transplantation. Here, we have optimized and characterized an immunomagnetic bead (IB) based cell sorting method to enrich CD34+CD38- cells from human bone marrow (BM). Our results suggest we can use clinically available technology (IB-based cell sorting) to achieve a ~ 10-fold reduction in vector requirements while still retaining the HSC required for clinical benefit after transplant.

[0090] We first used competitive transplant studies to determine the SCID repopulating activity of different subpopulations of BM CD34+ cells after 2-day ex-vivo culture and LV transduction. The CD34+ BM cells were sorted into 3 intervals of increasing CD38 expression, each marked with a distinct fluorescent LV vector, and competitively transplanted into NSG mice. At >16 weeks post-transplant, >90% of all hCD45+hCD34+ cells in NSG marrow were derived from the lowest -6% of CD38 negativity. [0091] We next optimized small-scale (50-100 mL of BM) enrichment of

CD34+CD38- cells using an IB-based cell isolation method. CD38+ cells were first depleted, and CD34+38- cells were subsequently selected. This double-step

CD34+CD38- purification non-specifically enriched granulocytes, which represented up to 50% of total isolated cells. Thus, we modified the first step of our protocol to include co-depletion of both CD15+ myeloid cells and CD38+cells, which increased the purity of CD34+38- cells up to 2-fold. The dual IB-based CD34+CD38- purification and standard CD34+ purification were performed in parallel on 4 independent BM samples. Recovery of CD34+CD38-cells from starting material (MNCs) was assessed by each method. The single-step IB CD34+ purification recovered 91.8±6.5% (mean±SD) of CD34+CD38- cells while double-step IB CD34+CD38- purification recovered 72±1.4% of

CD34+CD38- cells. An additional 20±6% of CD34+CD38- cells remained in the CD38+ fraction. When compared to single-step IB CD34+ purification, CD34+CD38- two-step purification reduced the total number of isolated cells by a range of 7.6-17.8-fold. [0092] We next compared the long-term engraftment capabilities of IB-purified

CD34+ and CD34+CD38- cells transduced with a LV expressing mCitrine. CD34+ or CD34+CD38- cells purified from an equivalent volume of marrow were transplanted into NSG mice at limiting cell doses. Purified cells were transduced at the same cell density and vector concentration, with the CD34+CD38- cell grafts requiring 1 1.8-fold less LV than CD34+ cells. At 20 weeks, recipients of CD34+ or CD34+CD38- purified cells had similar levels of human chimerism indicating retention of engraftment capacity by the enriched CD34+CD38- cells. Additionally, hCD45+ cells in engrafted NSG mice exhibited similar levels of mCitrine expression (MFI), suggesting that IB enrichment of CD34+CD38- cells does not affect gene transfer into HSC. [0093] One potential disadvantage of using purified CD34+CD38- cells in a clinical setting is delayed myeloid recovery in the post-transplant period. NSG mice transplanted with purified CD34+CD38- cells demonstrated reduced circulating human myeloid cells at 3 weeks post-transplant as compared to mice transplanted with CD34+ cells. In order to overcome this potential clinical hurdle, we explored a strategy of adding back non-transduced CD38+cells, which restored early levels of circulating human myeloid cells. We are currently investigating the effects of adding non-transduced CD38+ cells on long-term engraftment of gene-marked HSC.

[0094] In summary, we have developed a clinically applicable method to enrich

HSCs and reduce vector requirements. This strategy could directly contribute to clinical trials for hemoglobinopathies by addressing the factors currently limiting gene therapy methods.

Example 2

Transduction efficiency in CD34+38- vs 34+ cells, CB, MACS

Before starting: [0095] ] Make MACS buffer (500 mL PBS + 10.2 mL HAS (25%) + 2.05 mL EDTA (0.5M))

[0096] Make Retronectin coated plates [0097] Make x— vivo media

Day 1

[0098] Get fresh MNC from CB

[0099] Count cells: 82.5 x 105, contains a visible amount of RBCs in pellet, even after ficoll

[0100] Remove 4.2 x 106 cells for analysis

[0101] 1 tube of 4 x 106 cells (A) CI34 and CD38

[0102] 50,000 cells (A) C034 only

[0103] 50,000 cells (A) CD38 only

[0104] 50,000 cells for unstained

[0105] 50,000 cells for DAPI only

Bead separation— Deplete 38+ cells,,use LD column

[0106] 1) Centrifuge cells at 500 xg for 5 min. Remove media

[0107] 2) Resuspend in 160 uL PBS/2% FBS

[0108] 3) Add 40 uL CD38-PE. Stain at 4C for 30 min in fridge

[0109] 4) Add 2 ml. buffer, centrifuge at 500xg for 5 min, remove media

[0110] 5) Set aside small sample (2 μΐ.) for FACS (evaluate scaled up staining)

(B)

[0111] 6) Add 80 μΐ. buffer and 20 μΐ, anti-PE beads per 10 ⁷ cells, (640ul. buffer

+ 160 uL PE beads). Stain for 15 min in fridge

[0112] 7) Add 2 ml. buffer, centrifuge at 300 xg for 10 min, remove media

[0113] 8) Resuspend in 500 μΐ. buffer

[0114] 9) Use LD column, place in magnetic field

[0115] 10) Wash column with 2 ml. buffer

[0116] 11) Add 500 μΐ, of labeled cells to column (collect 38-)

[0117] 12) Wash column (and collect 318-) with 2x1 mL buffer (only add new buffer when column is empty) [0118] 13) Remove column from magnetic field, add lml. wash buffer and flush cells with syringe (collect 38+ cells) -Count collected cells:

[0119] 38-: 50.8 x 10 ⁶

[0120] 38+: 9.5 x 10 ⁶

[0121] Set aside 50,000 cells of each for FACS (C) = 38- cells, (D) = 38+ cells

Bead separation - Select 34+ cells, use MS column

[0122] 1) Centrifuge CD38-depleted cells at 500xg for 5 min'. Remove media

[0123] 2) Resuspend in 475 uL PBS/2% FBS

[0124] 3) Add 25 uL CD34— APC. Stain at 4C for 30 min in fridge

[0125] 4) Add 2 mL buffer, centrifuge at SOOxg for 5 min, remove media

[0126] 5) Resuspend in 80ul buffer/107 cells (475 uL) Set aside small sample (5 uL) for FACS (evaluate scaled up staining) (E)

[0127] 6) Add 20 ul. anti-APC beads per 107 cells (100 uL), stain for 15 min in fridge

[0128] 7) Add 2 mL buffer, centrifuge at: 300xg for 10 min, remove media

[0129] 8) Resuspend in 500 μΐ _^ buffer

[0130] 9) Use LS column, place column in magnetic field

[0131] 10) Add 3mL buffer to column to Wash

[0132] 11) Add cells in 500 buffer (collect 34-)

[0133] 12) Wash column 3x with 3mL UL. (collect 34~)

[0134] 13) Remove column from magnetic field, add 5 mL buffer and flush cells out with syringe (collect 34+)

[0135] Count collected cells:

[0136] 38-34+: 250,000

[0137] 38-34-: 44.1 x 10 ⁵

[0138] Set aside cells of each for FACES (F) = 3884- cells (took aliquot), (G) =

38-34+ cells (used all collected cells) FACS

[0139] A = total CB before separation— CD34— APC and CD38-PE

[0140] A = total CB before separation— CD34— APC

[0141] A 2 total CB before separation— CD38— PE [0142] B = Sample of scaled up staining for CD38— 'add no antibody, measure

[0143] C = 38- cells from column— add no antibody, measure

[0144] D = 38+ cells from column— add no antibody, measure

[0145] E = Sample of scaled up staining for CD34— add no antibody, measure

[0146] F = 38-34- cells from column, add no Ab, measure [0147] G : 38-34+ cells from column, add no Ab, measure

[0148] Used beads for compensation

[0149] See Figure 1.

Example 3

[0150] Lentiviral (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases.

Currently, HSC are enriched using immunomagnetic beads (IB) to select CD34 ⁺ cells prior to gene modification and transplantation. This strategy may be inefficient as the majority of CD34 ⁺ cells are short-term progenitors with a limited post-transplant lifespan. Here, we hypothesized that less LV per patient could be used by purifying CD34 ⁺ CD38 ^" cells, a population further enriched for HSC. Competitive transplant studies in immune-deficient mice revealed that >90% of long-term repopulating activity in human bone marrow (BM) CD34 ⁺ cells is contained within the lowest 6% of CD38 negativity. We next optimized a clinically relevant, IB-based method to purify

CD34 ⁺ CD38 ^" cells from human BM and mobilized peripheral blood (mPB) and achieved high recovery of CD34 ⁺ CD38 ^" cells with a ~10-fold reduction in total cell number. IB- purification of CD34 ⁺ CD38 ^" cells enriched SCID repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 ⁺ purification and did not affect gene marking of long-term HSC. CD34 ⁺ CD38 ^" cells showed delayed myeloid reconstitution which could be rescued by the addition of non-transduced CD38 ⁺ cells. LV transduction of CD34 ⁺ CD38 ^" cells coupled with co-transplantation with non-transduced CD38 ⁺ cells achieved long-term gene-marked engraftment comparable to modification of bulk CD34 ⁺ cells, while utilizing ~7-fold less LV. Thus, we demonstrate a novel method to improve gene therapy efficiency while maintaining clinical efficacy.

Materials and Methods.

Mononuclear Cell (MNC) isolation

[0151] Healthy adult BM and mPB were obtained from commercial sources (All

Cells, Hemacare). Umbilical CB was obtained after vaginal and cesarean deliveries at UCLA Medical Center. All specimens obtained have been deemed as anonymous medical waste exempt from IRB review. MNCs were isolated using Ficoll-Paque PLUS (GE Healthcare) density centrifugation within 48 hours of collection. IB purification of CD34 ⁺ and CD34 ⁺ 38 ^' cells

[0152] All microbeads and magnetic columns were purchased from Miltenyi

Biotech. Prior to column separation, MNCs were stained with CD38-PE (1 : 15, Clone IB6, Miltneyi) CD34-FITC (1 : 10, Clone 581, Becton-Dickinson Biosciences [BD]), and CD45-APC (1 : 10, Clone HI30, BD) for 30 minutes at 4°C. Stained MNCs were washed once with flow buffer (phosphate buffered saline (PBS)/0.5% Bovine Serum Albumin (BSA)/2mM EDTA) and purified by either CD34 ⁺ or CD34 ⁺ CD38 ^" IB purification. To purify CD34 ⁺ cells, MNCs were incubated with anti-CD34 microbeads (1 :5) at 4°C for 30 minutes, washed, and purified on an LS column. CD34 D38 ^" cells were purified by first incubating MNCs with anti-PE microbeads (1 :5 -1 :25) and anti-CD15 microbeads (1 :5) for 15 min at 4C. Cells were washed and separated on an LD column. The

CD387CD15 ⁺ fraction was flushed from the column. The CD387CD15 ^" fraction was collected, washed, and subsequently selected with anti-CD34 microbeads. To obtain absolute CD34 ⁺ CD38 ^" cell counts in each fraction, 3 x 100 μΐ. aliquots were added to 300 μΐ. of flow buffer and 50 μΐ. of counting beads (eBioscience) and DAPI (1 : 1000), and analyzed on a LSRII or LSR Fortessa flow cytometer (BD Biosciences), (see, e.g., Figure 11). Table 1 shows total cell counts by IB purified fraction.

Table 1. Total cell counts by IB purified fraction. Numbers show total cells isolated in each fraction, with CD34+CD38- cell counts listed in parentheses.

(102,668) (84,718) (62,910) (22,241)

7.9 x 10 ⁶ 5.5 x 10 ⁵ 4.6 x 10 ⁴ 5.9 x 10 ⁶

BM #1

(27,730) (26,458) (18,665) (4,789)

3.8 x 10 ⁶ 6.1 x 10 ⁵ 7.1 x 10 ⁴ 6.92 x 10 ⁶

BM #1

(37,842) (36,194) (23,649) (2,448)

1.5 x 10 ⁷ 4.3 x 10 ⁵ 7.0 x 10 ⁴ 1.1 x 10 ⁷

BM #1

(36,755) (28,148) (21, 145) (6,220)

2.8 x 10 ⁷ 5,6 x 10 ⁵ 6.2 x 10 ⁴ 2.1 x 10 ⁷

mPB#l

(38,411) (32,145) (28,849) (4,557)

1.9 x 10 ⁷ 4.8 x 10 ⁵ 3.2 x 10 ⁴ 1.9 x 10 ⁷

mPB#2

(30,746) (27,023) (17,979) (18,861)

1.2 x 10 ⁷ 5.4 x 10 ⁵ 4.3 x 10 ⁴ 5.7 x 10 ⁷

mPB#3

(10,421) (9,510) (9,692) (400)

[0153] In some xenograft experiments, CD3-depleted MNCs were used as a starting material. MNCs were incubated with anti-CD3 microbeads (1 :5) at 4°C for 15 minutes, washed, and separated on an LD column. CD3 ^" cells were immediately processed by further CD34 ⁺ or CD34 ⁺ CD38 ^" IB purification. FACS

[0154] BM MNCs were enriched for CD34 ⁺ cells as described and stained with

CD38-PE (ΊΒ6, Miltenyi) and CD34-APC (BD) in flow buffer for 30 minutes at 4°C, followed by washing to remove unbound antibody. DAPI (1 : 1000) was added just before analysis. FACS Aria II (BD), sort CD34 ⁺ cells into defined intervals of CD38 expression. Cells were gated on viable, single CD34 ⁺ cells (Figure 10A) prior to defining CD38 intervals.

LV transduction

[0155] Construction, packaging, and titering of CCL-Ubiq-mCitrine-PRE-FB-

2XUSE, CCL-Ubiq-mStrawberry-PRE-FB-2XUSE, and CCL-Ubiq-mCerulean-PRE- FB-2XUSE have been described (Baldwin et al. (2015) Stem Cells. 33(5): 1532-1542). Cells were plated in retronectin (Takara) coated plates (20 μg/ml) at a density of 0.5- lxlO ⁶ cells/mL in X-VIV015 medium (Lonza) containing lx

glutamine/penicillin/streptomycin (Gemini), 50 ng/mL stem cell factor (SCF), 50 ng/mL fms-related tyrosine kinase 3 ligand (Flt3-L), 50 ng/mL thrombopoietin (TPO), and 20 ng/mL interleukin 3 (IL-3) (Peprotech). LV was added to a concentration of 2xl0 ⁷

TU/mL and incubated with cells for 24 hours prior to transplant. All LV concentrations (TU/mL), cell concentrations (cells/mL), and cell densities (cells/cm ² ) were kept constant during transduction of CD34 ⁺ and CD34 ⁺ CD38 ^" cells.

Xenografts

[0156] 6-10 week old male and female NOD.Cg-Prkdsci-dI12rgtmlWjil/SzJ (NSG) and NOD.Cg-PrkdcscidI12rgtmlWjlTg(CMV-IL3,CSF2,KITLG)lEav/MloySz J (NSG-SGM3) mice (Jackson Laboratory) were used as transplant recipients. Mice were irradiated 4-6 hours prior to transplantation using a ¹³⁷ Cesium source at a total dose of 250 Rads (-101 Rads/min). Cells were resuspended in PBS and administered via i.v. injection into the retro-orbital sinus. CD34 ^" cells were irradiated (10Gy) and added to grafts as filler cells (Figure 14, table).

Engraftment analysis

[0157] Engraftment of human cells in PB, BM, and spleen was evaluated by flow cytometry using anti-human CD45-APC (HI30), anti-murine CD45-PE (Clone 30-FII), anti-human CD33-V450 (Clone P67-6), anti-human CD66b-V450 (Clone G10F5), anti- human CD15-V450 (Clone HI98), anti-human CD3-PerCP-Cy5.5 (Clone SK7), anti- human CD19-APC-Cy7 (SJ25CI), and anti-human CD34-PE-Cy7 (Clone 581) (all antibodies BD).

Determination of vector copies (VC) per human cell

[0158] The average VC/human cell was measured in engrafted NSG BM samples as previously described (Baldwin et al. (2015) Stem Cells. 33(5): 1532-1542). Briefly, LV DNA content was quantified using a digital droplet PCR probe specific to the HIV-1 Psi region and normalized to the autosomal human gene SDC4 gene.

Statistical analyses

[0159] Pairwise comparison was performed by unpaired t test within the framework of one-way ANOVA. Two-group comparisons by Wilcoxon rank sum test was performed when the assumption of normality was not met. Hypothesis testing was two-sided, and a significance threshold 0.05 for p-value was used. Limiting dilution analysis was performed using online software provided by WEHI bioinformatics (Hu and Smyth (2009) J. Immunol. Meth. 347(1): 70-78). Results

>90% of CD34 ⁺ long-term repopulating activity is contained within the lowest 6% of CD38 negativity

[0160] In order to identify a target cell population to purify for gene therapy, we first sought to determine which subsets of BM CD34 ⁺ cells are capable of long-term engraftment after ex vivo culture and LV transduction. Here, we performed a

competitive transplant assay in immune-deficient NSG mice utilizing CD34 ⁺ cells with varying levels of CD38 expression (Figure 3 A). Freshly isolated, CD34-enriched BM cells were FACS sorted into three different cell populations with increasing levels of CD38 expression. Each population of sorted cells was transduced with one of 3 fluorescent LV: CCL-UBC-mCitrine, CCL-UBC-mStrawbeny, or CCL-UBC- mCerulean. Following transduction, equal numbers of cells from each of three differentially labeled populations were competitively co-transplanted into sub-lethally irradiated NSG mice. Fluorescent reporter gene color choice for each sorted fraction was rotated to account for potential differences in transduction or detection efficiency among vectors (Figure 10D).

[0161] At 16-18 weeks post-transplantation, engrafted hCD45 ⁺ cells in NSG BM were analyzed by flow cytometry for mCitrine, mStrawberry, and mCerulean expression to determine the long-term engraftment potential of each initially sorted population. Two separate transplants were performed using different intervals of CD38 expression (Figure 3B). In transplant #1, >90% of hCD45 ⁺ cells present in the bone marrow were derived from CD34 ⁺ CD38° ^"6% cells, while <10% of hCD45 ⁺ cells were derived from CD34 ⁺ CD38 ^6"12% and CD34 ⁺ CD38 ^12"18% cells. In transplant #2, 54% of hCD45 ⁺ cells were derived from CD34 ⁺ CD38° ^"3% cells, with an additional 40% of hCD45 ⁺ cells derived from the CD34 ⁺ CD38 ^3"6% fraction. For each transplant, the observed patterns were consistent in myeloid (CD33 ⁺ ), lymphoid (CD19 ⁺ ), and HSPC (CD34 ⁺ ) lineages.

[0162] Collectively, these results suggest that after ex vivo culture and LV transduction, the majority of long-term repopulating activity in CD34 ⁺ adult bone marrow cells is contained within the lowest 6% of CD38 negativity. We therefore used this cutoff to phenotypically define "CD34 ⁺ CD38 ^" cells" for our subsequent experiments. Titration of CD38 magnetic labeling optimizes recovery and purity of CD34 ⁺ CD38 ^" cells

[0163] We next sought to optimize a clinically relevant, IB-based method to purify CD34 ⁺ CD38 ^" cells. We chose to first deplete CD38 ⁺ cells and subsequently select CD34 ⁺ cells. CD38 magnetic labeling was performed using a primary anti-CD38-PE conjugated antibody followed by incubation with anti-PE magnetic beads. Unlabeled CD38 ^" cells can be relabeled with CD34 beads, thereby preventing the necessity of a bead removal step. CD34 ⁺ cells exhibit a gradient pattern of CD38 expression rather than discrete positive and negative populations. We found that the proportion of cells separated into CD38 ^" and CD38 ⁺ fractions could be adjusted by varying the intensity of CD38 magnetic labeling with different concentrations of magnetic beads (Figures 4 A and 4B). MNCs from CB, BM, and mPB were separated into CD38 ^" and CD38 ⁺ fractions using three different concentrations of anti-PE beads. Strong magnetic labeling (anti-PE beads 1 :5) greatly enriched for CD34+CD38- cells (13-, 16-, and 15-fold for CB, BM, and mPB respectively), but resulted in low recovery of CD34 ⁺ CD38 ^" cells (50% 19%, and 59% for CB, BM, and mPB). Weak magnetic labeling (anti-PE beads 1 :25) resulted in high recovery of CD34 ⁺ CD38 ^" cells (93%, 67%, and 96% for CB, BM, and mPB) but reduced enrichment of CD34 ⁺ CD38 ^" cells (4.6-, 10.3-, and 4.7-fold for CB, BM, and mPB). Thus, there is a tradeoff between recovery and fold-enrichment of CD34 D38 ^" cells that can be optimized by altering the strength of CD38 magnetic labeling. Here, we observe that anti-PE bead concentrations of 1 : 10 for mPB and >1 :25 for BM can optimize both enrichment and recovery of CD34 ⁺ CD38 ^" cells.

Co-depletion of CD15 ⁺ and CD38 ⁺ cells leads to greater CD34 ⁺ CD38 ^' purity

[0164] In BM and CB, we observed that CD38 depletion followed by CD34 selection led to a final cell product with <50% CD34 ⁺ cells due to contamination of CD15 ⁺ granulocytes (Figure 5 A, top). Similar absolute numbers of granulocytes were isolated by CD34 ⁺ or CD34 ⁺ CD38 ^" purification (Figure 5B) suggesting that the CD38 depletion step does not increase non-specific granulocyte selection. To obtain more highly purified CD34 D38 ^" cells, we included a CD 15 magnetic bead during the CD38 depletion step, allowing us to simultaneously co-deplete CD38 ⁺ cells and CD15 ⁺ granulocytes prior to CD34 selection (Figure 5C). In BM, addition of a CD 15 bead increased purity of CD34 ⁺ CD38 ^" cells from 24.5% of total cells to 70.5% of total cells, thus further reducing total number of cells isolated prior to transduction. Based on these findings, all subsequent CD34 ⁺ CD38 ^" purifications were performed using CD38/CD15 co-depletion.

Optimized CD34 ⁺ CD38 ^' dual IB purification enriches CD34 ⁺ CD38 ^' cells with high recovery and results in efficient HSC gene-marking

[0165] We next compared the optimized CD34 ⁺ CD38 ^" IB purification strategy to standard CD34 ⁺ selection in multiple BM and mPB samples from healthy donors. For each sample analyzed, two identical fractions of MNCs were purified by either CD34 ⁺ single-step IB purification or CD34 ⁺ CD38 ^" dual IB purification (Figures 6A, 6B). In BM, CD34 ⁺ CD38 ^" dual IB purification of starting MNCs recovered a median

62.5%(range 57.2-65.6%) of phenotypically defined CD34 ⁺ CD38 ^" cells, whereas standard CD34 selection recovered a median of 93.1%(range 80-95.6%) of CD34 ⁺ CD38 ^" cells. CD34 ⁺ CD38 ^" dual IB purification enriched for CD34 ⁺ CD38 ^" cells an additional 8.7-fold (range 4.8-12) beyond standard CD34+ purification. Similar results were achieved in mPB, with a median CD34 ⁺ CD38 ^" recovery of 75.1%(range 58.4-91.0%) by dual CD34 ⁺ CD38 ^" purification, and 87.9%(range 83.7-93.2%) by standard CD34 selection. CD34 ⁺ CD38 ^" dual IB purification enriched for CD34 ⁺ CD38 ^" cells an additional 12-fold (range 8.7-13.2) beyond standard CD34 ⁺ purification.

[0166] In order to assess functional HSC content in each purified population, we performed a SCID repopulating cell (SRC) assay. Limiting dilution analysis of CCL- UBC-mCitrine LV-transduced cells demonstrated a 12-fold enrichment of SRC frequency within CD34 ⁺ CD38 ^" cells (1 in 2,314 cells; 95% confidence interval: 1/1,241- 1/4,314) compared to CD34 ⁺ cells (1 in 28,248 cells; 95% confidence interval: 1/14,701- 1/54,278) (Figure 6C). Analysis of long-term (>16 week) engrafted hCD45 ⁺ cells in NSG BM revealed no significant difference in average vector copy number per human cell between groups, suggesting that the dual CD34 ⁺ CD38 ^" IB purification method allows for efficient transduction of HSC while using less cells and LV (Figure 6D).

CD34 ⁺ CD38 ^" cells show delayed myeloid recovery that can be rescued by increasing cell dose or adding back non-transduced CD38 ⁺ cells

[0167] Purifying CD34 ⁺ CD38 ^" cells may provide the advantage of utilizing less LV to transduce HSC. However, transplanting a population of quiescent, stem-enriched cells may cause delayed myeloid recovery. Therefore, we compared the early myeloid potential of IB-purified CD34 ⁺ and CD34 ⁺ CD38 ^" cells. Here, we transplanted purified cells into the NSG-SGM3 mouse model (Miller et al. (2013) Blood, 121(5): el-e4), an immune-deficient mouse model which supports human myelopoiesis through

constitutive expression of human cytokines IL3, GM-CSF, and SCF. In order to reflect a clinically relevant scenario, transplanted cell doses for each group represented the total cells obtained from IB purification of a standard volume of marrow (marrow equivalent or ME). 1ME of CD34 ⁺ CD38 ^" cells showed decreased early production of circulating human myeloid cells as compared to lME of CD34 ⁺ cells (Figure 7A). 2.5 ME of CD34 ⁺ CD38 ^" cells achieved myeloid reconstitution comparable to 1 ME of CD34 ⁺ cells while 6ME of CD34 ⁺ CD38 ^" cells resulted in myeloid reconstitution ~3-fold higher than 1 ME of CD34 ⁺ cells. These results demonstrate that dual IB-purified CD34 ⁺ CD38 ^" cells are capable of early myeloid reconstitution, but contain less early myeloid potential than CD34 ⁺ cells purified from an equivalent volume of BM.

[0168] We next investigated if co-transplanting non-transduced CD38 ⁺ cells

(obtained during IB depletion of MNCs) alongside transduced CD34 ⁺ CD38 ^" cells could produce early hematopoietic recovery comparable to CD34 ⁺ cells. In order to avoid xenogeneic GVHD in recipient mice receiving CD38 ⁺ cells, we purified CD34 ⁺ ,

CD34 ⁺ CD38 ^" and CD38 ⁺ populations from CD3-depleted BM MNCs (Figure 7B). IB purified CD34 ⁺ or CD34 ⁺ CD38 ^" cells were transduced with CCL-UBC-mCitrine LV, while CD38 cells were cultured in parallel transduction conditions without LV. NSG- SGM3 mice received lME of the grafts depicted in Figure 7B: 1. CD34 ⁺ cells

(+mCitrine LV), 2. CD34 ⁺ CD38 ^" cells (+mCitrine LV) 3. CD34 ⁺ CD38 ^" cells (+mCitrine LV) + CD38 ⁺ cells (non-transduced) or 4. CD38 ⁺ cells (non-transduced). Irradiated CD34 ^" cells were added as needed so that each mouse received the same number of total cells (Fig. 14 table).

[0169] Two transplants were performed using BM from independent donors. Transplant #1 used highly purified CD34 ⁺ CD38 ^" cells (-1% of CD34 ⁺ cells), and transplant #2 used more moderately purified CD34 ⁺ CD38 ^" cells (-12% of CD34+ cells) (Figure 7C). In transplant #1, mice receiving CD34 ⁺ CD38 ^" cells showed significantly reduced levels of circulating human neutrophils at 3 weeks compared to mice

transplanted with CD34 ⁺ cells. The addition of non-transduced CD38 ⁺ cells to the graft restored circulating neutrophils levels back to the levels achieved by CD34 ⁺ transplant. The same pattern was observed in transplant #2 but did not reach significance when using more moderately purified CD34 ⁺ CD38 ^" cells. In both transplants, the majority of human neutrophils in arm #3 were mCitrine ^" , confirming that these cells derive from non-transduced CD38 ⁺ cells. In the NSG model, we observed a similar defect in early myelopoiesis from transplanted CD34 ⁺ CD38 ^" cells and rescue by the add-back of CD38 ⁺ cells (Figure 13), suggesting that these results are not unique to the NSG-SGM3 model. Collectively, these data show that addition of cultured, non-transduced CD38 ⁺ cells can restore early myeloid engraftment when added to transduced CD34 ⁺ CD38 ^" cells. Addition of CD38 ⁺ cells enhances long-term engraftment of gene-modified

HSC

[0170] We next assessed the effect of adding back non-transduced CD38 ⁺ cells on long-term gene-marked engraftment. Here, we transplanted IB-purified CD34 ⁺ , CD34 D38 ^" , and CD38 ⁺ cells into NSG mice using the same 4 experimental arms depicted in Figure 7B. At 16 weeks post-transplant, recipients of CD34 ⁺ CD38 ^" cells showed decreased engraftment of total (hCD45 ⁺ ) and gene-marked (mCitrine ⁺ hCD45 ⁺ ) cells as compared to recipients of CD34 ⁺ cells (Figure 8A). Addition of non-transduced CD38 ⁺ cells to the graft of transduced CD34 ⁺ CD38 ^" cells significantly increased engraftment of gene-marked (hCD45 ⁺ mCitrine ⁺ ) cells to levels achieved by CD34 ⁺ cell transplants. Importantly, this was accomplished using 6.2 and 7.8-fold less LV (in 2 independent experiments) compared to the volumes required to transduce bulk CD34 ⁺ cells. All transplanted arms showed similar lineage distribution, with the exception of mice transplanted with CD38 ⁺ cells alone which showed a significant myeloid bias in engrafted cells (Figure 8B). PB analysis showed comparable engraftment of total hCD45 ⁺ cells at all time points after transplant of transduced CD34 ⁺ cells or transduced CD34 ⁺ CD38 ^" cells co-transplanted with non-transduced CD38 ⁺ cells (figure 6c, top). At early time points (3-12 weeks), transduced CD34 ⁺ cells showed higher levels of gene- marked (mCitrine+) engraftment than transduced CD34 ⁺ CD38 ^" cells co-transplanted with non-transduced CD38 ⁺ cells (Figure 8C, bottom). However, after 12 weeks, we observed equivalent levels of gene-marked engraftment in these two groups. These data suggest that non-transduced CD38 ⁺ cells drive early hematopoiesis (3-12 weeks), while gene- marked CD34 ⁺ CD38 ^" cells take over hematopoiesis 12 weeks after transplant.

Discussion

[0171] We have demonstrated a clinically relevant, IB-based method for purifying CD34 ⁺ CD38 ^" cells from CB, mPB and BM. We further show that modification of CD34 ⁺ CD38 ^" cells with a reduced LV dose coupled with co-transplantation with non- modified CD38 ⁺ cells produced early myeloid reconstitution and long-term engraftment of gene-marked cells comparable to traditional methods of modifying bulk CD34 ⁺ cells. The strategy explored here is broadly applicable to a number of HSC-based gene therapy applications, and may be especially useful in gene therapy for hemoglobinopathies, where clinical scale LV production has proven difficult due to the large size and complexity of the human ?-globin gene expression cassette. [0172] While a large body of work has characterized cell surface markers and repopulation capacity of human HSC, much of this work has been performed using FACS-purified CB. CB is a widely available and affordable source of stem cells for research use; however the majority of clinical transplants are performed using adult sources of stem cells such as BM and mPB. Importantly, our work has explored IB purification of clinically relevant sources of stem cells, which may allow rapid translation of our findings to current clinical gene therapy trials.

[0173] While a number of in vivo repopulating studies have shown that HSC are enriched within the CD34 ⁺ CD38 ^" fraction (Larochelle et al. (1996) Nat Med. 2(12): 1329-1337; Bhatia et a/. (1997) Proc. Natl. Acad. Sci. USA, 94(10): 5320-5325; Hogan et al. (2002) Proc. Natl. Acad. Sci. USA, 99(1): 413-418; Ishikawa et al. (2003)

Leukemia, 17(5): 960-964; McKenzie et al. (2007) Exp. Hematol. 35(9): 1429-1436), most of these studies have evaluated uncultured cells, whereas ex vivo culture/LV- transduction likely alters repopulating capacity. Additionally, prior work has used variable definitions for CD38 negativity ranging from the lowest 1% to 30% of CD38 expression. Some work has also suggested that a CD34 ⁺ CD38 ^low fraction contains long- term repopulating activity (Hogan et al. (2002) Proc. Natl. Acad. Sci. USA, 99(1): 413- 418; McKenzie et al. (2007) Exp. Hematol. 35(9): 1429-1436). Here, we find that within freshly isolated BM CD34 ⁺ cells, only the lowest 6% of CD38 negativity is capable of long-term NSG repopulation after LV transduction/ex vivo culture. Thus purifying CD34 cells based on CD38 expression alone could lead to a theoretical -16 fold enrichment of long-term repopulating activity.

[0174] High individual donor variability in CD38 expression could be a potential hurdle to clinical translation. Because the lowest 6% of CD38 negativity may not encompass >90% of HSC for every individual, a conservative cut-off for CD38 negativity may increase the chance of retaining HSC. Our results show that CD38 negativity can be adjusted by titration of magnetic beads, thereby achieving optimal recovery of CD34 ⁺ CD38 ^" cells while also maximizing HSC enrichment. Clinical scale- up to GMP-grade IB sorting systems will likely require additional titration of CD38 magnetic labeling specific to the system parameters (magnetic field strength, column flow rate, etc.).

[0175] In the dual CD34 ⁺ CD38 ^" IB protocol, we found that CD38 ⁺ /CD15 ⁺ co- depletion increased purity of CD34 ⁺ CD38 ^" cells by preventing the non-specific enrichment of CD15 ⁺ granulocytes. Due to the relative rarity of CD34 ⁺ CD38 ^" cells (-0.1-0.3% of MNCs), non-specific enrichment of granulocytes can represent >50% of total cells after CD34 ⁺ CD38 ^" purification. In contrast, CD34 ⁺ cells are -10 times more abundant than CD34 ⁺ CD38 ^" cells (-1-3% of MNCs), thus the relative contribution of granulocytes is minor in CD34 ⁺ purified cells (<10%). As clinical vector doses are calculated on a per cell basis, contaminating granulocytes present in CD34 ⁺ CD38 ^" purified populations could reduce vector MOI and limit the vector dose reduction achieved by CD34 ⁺ CD38 ^" IB purification. Furthermore, cryopreservation of a granulocyte-rich cell population could potentially lead to poor cell recovery upon thawing due to granulocyte death/clumping. [0176] Application of the optimized CD34 ⁺ CD38 ^" dual IB method to samples of healthy BM and mPB achieved -75% recovery and -10-fold enrichment of

phenotypically defined CD34 ⁺ CD38 ^" cells in mPB, and -60% recovery and ~5-10-fold enrichment in BM. In BM, CD34 ⁺ CD38 ^" purification results in a 12-fold enrichment of SRC beyond standard CD34 ⁺ selection. We observed no difference in transduction efficiency of IB purified CD34 ⁺ CD38 ^" or CD34 ⁺ cells when evaluated by VCN analysis of long-term NSG-engrafted human cells. This is in contrast to our lab's previous demonstration that highly purified (FACS-sorted) CB CD34 ⁺ CD38 ^" cells demonstrate enhanced transduction in vitro compared to CD34 ⁺ cells (Baldwin et al. (2015) Stem Cells. 33(5): 1532-1542). This discrepancy could be due to differences in the tissue analyzed (BM vs. CB), CD34 ⁺ CD38 ^" purity (moderately purified IB-sorted cells vs highly purified FACS-sorted cells) or post-transduction assay {in vitro culture vs. in vivo xenografts). Importantly, our results here suggest that the dual IB CD34 ⁺ CD38 ^" purification does not impede LV transduction of long-term HSC.

[0177] We next explored the early myeloid potential of IB purified CD34 ⁺ CD38 ^" cells. Prior work has shown that short-term repopulating activity is enriched in the

CD34 ⁺ CD38 ⁺ fraction (Glimm et al. (2001) J. Clin. Invest. 107(2): 199-206) suggesting that CD34 ⁺ CD38 ⁺ progenitors may be necessary for early myeloid recovery. However, murine studies have suggested that high doses of purified stem cells can lead to rapid hematopoietic recovery (Uchida et al. (1994) Blood, 83(12): 3758-3779; Uchida et al. (1998) J. Clin. Invest. 101(5): 961-966). Clinical trials using FACS-sorted CD34 ⁺ CD90 ⁺ cells have demonstrated prompt neutrophil and platelet engraftment in patients transplanted with >0.8 x 10 ⁶ CD34 ⁺ CD90 ⁺ cells/kg (Tricot et al. (1988) Blood, 91(12): 4489-4495; Negrin et al. (2000) Biol. Blood Marrow Transpl. 6(3): 262-271; Michallet et al. (2000) Exp. Hematol. 28(7): 858-870). However, the purified CD34 ⁺ CD90 ⁺ populations in these studies represented -50% of total CD34 ⁺ cells, whereas the IB- purified CD34 ⁺ CD38 ^" cells evaluated here represent -10% of total CD34 ⁺ cells.

Therefore, it is largely unknown if high doses of purified CD34 ⁺ CD38 ^" cells could provide rapid myeloid reconstitution in a clinical transplant setting, or if co-transplant of CD34 ⁺ CD38 ⁺ cells will be required.

[0178] Here, we observed that IB purified CD34 ⁺ CD38 ^" cells were capable of early myeloid reconstitution, but contained less early myeloid potential than an equivalent marrow volume of CD34 ⁺ cells. Our data suggest that -2-3 ME of IB purified CD34 ⁺ CD38 ^" cells may be required to achieve early myeloid reconstitution comparable to 1 ME of CD34 ⁺ cells. In a clinical scenario, it is possible that the total product of CD34 ⁺ CD38 ^" cells obtained will be above the threshold required for sufficient myeloid reconstitution. However, we favor the approach of adding non-transduced CD38 ⁺ cells (containing CD34 ⁺ CD38 ⁺ progenitors) to the graft, as transplant of bulk CD34 ⁺ cells in gene therapy clinical trials has historically provided prompt myeloid reconstitution.

[0179] In long-term transplant assays, we observed decreased engraftment of both total and gene-marked cells in recipients receiving CD34 ⁺ CD38 ^" cells alone as compared to recipients receiving CD34 ⁺ cells. However, when non-transduced CD38 ⁺ cells were added to transduced CD34 ⁺ CD38 ^" cells, levels of gene-modified engraftment were equivalent to those achieved by standard CD34 ⁺ transduction/transplant. These data suggest that the CD34 ⁺ CD38 ^" dual IB purification method efficiently recovers HSC, but requires co-transplantation of CD34 ⁺ CD38 ⁺ cells for optimal long-term engraftment. In agreement with our findings, prior work has demonstrated that cycling CD34 ⁺ CD38 ⁺ cells facilitate HSC engraftment (Verstegen et al. (1998) Blood, 91(6): 1966-1976;

Bonnet (1999) Bone Marrow Transplant, 23(3): 203-209; Byk et al. (2005) Stem Cells, 23(4): 561-574) through enhancement of SDF-l-mediated homing, and secretion of metalloproteinase (MMP)-9 (Byk et al. (2005) Stem Cells, 23(4): 561-574). [0180] In our CD38 add-back transplant studies, we used a conservative CD38 depletion strategy (6.2-7.8-fold reduction in cell number/LV) in order to minimize the number of HSC retained in the CD38 ⁺ fraction. Further reductions in total cell number and LV dose could be achieved using a more stringent CD38 depletion. However, this approach may leave greater numbers of non-transduced HSC in the CD38 ⁺ fraction which could compete for limited niche space and reduce overall gene-marked engraftment. An alternative strategy is expansion of highly-purified, transduced CD34 ⁺ CD38 ^" cells with compounds such as SR-1 (Boitano et al. (2010 Science, 329(5997): 1345-1348; Wagner et al. (2016) Cell Stem Cell, 18(1): 144-155), PGE2 (North et al. (2007) Nature, 447(7147): 1007-1011 ; Cutler et al. (2013) Blood. 122(17): 3074-3081), or UM171 (Fares et al. (2014) Science, 345(6203): 1509-1512) to expand the progenitor compartment. Thus, expansion of highly-purified, transduced

CD34 ⁺ CD38 ^" cells could allow for prompt engraftment while avoiding any competitive disadvantage from non-transduced CD38 ⁺ cells. [0181] In summary, we demonstrate a method to improve the efficiency gene therapy for genetic blood cell diseases through improved HSC enrichment and reduced LV dose.

0182] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.