Compositions and methods for enhanced t cell reconstitution

Abstract

The present disclosure relates generally to engineered haematopoietic stem or progenitor cells (HSPCs) that enhance T cell reconstitution following haematopoietic stem cell transplantation (HSCT). In particular, the engineered HSPC of the present disclosure have been modified to increase expression of an X-linked lymphocyte-regulated (Xlr) gene or an ortholog thereof relative to an equivalent unmodified HSPC. The present disclosure also relates reagents and methods for engineering a HSPC, and the use of engineered HSPC for enhancing T cell reconstitution following HSCT, and in methods for the treatment of haematological diseases

Description

COMPOSITIONS AND METHODS FOR ENHANCED T CELL RECONSTITUTIONRelated applications

[0001] This application claims priority from Australian Provisional Patent Application No. 2025900307 filed on 5 February 2025, the entire content of which is incorporated by reference.Field

[0002] The present disclosure relates generally to engineered haematopoietic stem or progenitor cells (HSPCs) that enhance T cell reconstitution following haematopoietic stem cell transplantation (HSCT). In particular, the engineered HSPC of the present disclosure have been modified to increase expression of an X-linked lymphocyte-regulated (Xlr) gene or an ortholog thereof relative to an equivalent unmodified HSPC. The present disclosure also relates reagents and methods for engineering a HSPC, and the use of engineered HSPC for enhancing T cell reconstitution following HSCT, and in methods for the treatment of haematological diseases.Background

[0003] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0004] Haematopoietic stem cell transplantation (HSCT) is the most successful stem cell therapy providing a potentially curative strategy for >100,000 paediatric and adult patients worldwide each year. Notwithstanding, the leading cause of transplant-related morbidity and mortality remains infection due to a compromised immune system due to the preparative conditioning regime of chemotherapy and radiotherapy prior to the delivery of exogenous haematopoietic stem and progenitor cells (HSPC). Experimental research and clinical practice have demonstrated that total HSPC cell dose influences the time to reconstitution of innate immune cells particularly neutrophils. However, adaptive immunecell reconstitution, particularly of T cells, takes months following HSCT, which leaves patients susceptible to life threatening opportunistic infections.

[0005] The bone marrow provides a rich fabric of cellular interactions and signals to support HSPC maintenance, and the cues for commitment and differentiation of megakaryocyte, erythroid, myeloid and B lymphoid haematopoietic lineages. In contrast, the production of T cells represents a more elaborated developmental paradigm characterised by a number of molecularly distinct progenitor cells in the bone marrow having T lymphoid potential, rare thymic seeding progenitors (TSP) exiting the bone marrow and circulating to the thymus, and restrictive capacity of the thymic niche to accept TSPs and consequently, enforce T cell canalisation. The molecular foundations that underpin this process within HSPCs remains largely unknown. There remains, therefore, an urgent need to elucidate the cell-intrinsic transcriptional program in transplanted HSPCs that facilitates early thymic mediated T cell reconstitution with a view to develop clinically actionable reagents and methods to improve adaptive immune cell reconstitution following HSCT.Summary

[0006] The present disclosure is predicated, in part, on the surprising finding that expression of a X-linked lymphocyte-regulated (Xlr) gene or an ortholog thereof is characteristic of a cell-intrinsic transcriptional program in transplanted haematopoietic stem and progenitor cells (HSPCs) that facilitate early thymic mediated T cell reconstitution in patients with a compromised immune system. This finding has been reduced to practice by engineering HSPCs to ectopically express an Xlr gene or an ortholog thereof, resulting in HSPCs having a lymphoid bias favoring the differentiation to T cells relative to B cells. 10007] Accordingly, in one aspect, the present disclosure provides an engineered HSPC, which has been modified to ectopically express an Xlr gene or an ortholog thereof relative to an equivalent unmodified HSPC.

[0008] In another aspect, there is provided a composition comprising a population of the engineered HSPC disclosed herein.

[0009] In another aspect, there is provided nucleic acid construct comprising a nucleotide sequence encoding an XLR protein or an ortholog thereof.

[0010] In another aspect, there is provided a vector comprising the nucleic acid construct disclosed herein.

[0011] In another aspect, there is provided a method for engineering an HSPC to ectopically express anXlr gene or an ortholog thereof, wherein the method comprises:(a) contacting an isolated HSPC, or a population thereof, with the nucleic acid construct, or the vector disclosed herein; and(b) culturing the HSPC of step (a) in vitro or ex vivo for a time and under conditions suitable to ectopically express an Xlr gene or an ortholog thereof relative to an equivalent unmodified HSPC.

[0012] In another aspect, there is provided a method for enhancing T cell reconstitution following haematopoietic stem cell transplantation, the method comprising administering an effective amount of the engineered HSPC (e.g., a population of engineered HSPCs) or the composition disclosed herein to a subject in need thereof

[0013] In another aspect, there is provided a method for the treatment of a haematological disease, the method comprising administering the engineered HSPC (e.g., a population of engineered HSPCs) or the composition disclosed herein to a subject in need thereof

[0014] In another aspect, there is provided a use of the engineered HSPC (e.g., a population of engineered HSPCs) or the composition disclosed herein in the manufacture of a medicament for the treatment of a haematological disease.Brief Description of the Drawings

[0015] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the accompanying drawings.

[0016] Figure 1 shows that haematopoietic stem and progenitor cells (HSPC) undergo self-renewal and differentiation in PVA-bases expansion cultures. (A) A schematic representation of the experimental process for temporal analysis of ex vivo HSPC expansion.(B) A graphical representation of the number of cells in culture (y-axis) and time (x-axis).(C) A series of flow cytometry (FACS) contour plots showing the immunophenotypic composition of the culture over time when grown from sorted LSK or Lin. neg. cells. (D) Agraphical representation of UMAP projection of scRNA-seq from ex vivo cultured HSPCs. Leiden clustering was performed for a resolution of 0.7 and clusters were annotated based on the expression of established marker genes including Me com. Hlf Fit 3, Klfl, Fcgr3, Elane, Csflr and Cmal . (E-G) A series of graphical representations of UMAP projections with cell annotations indicates the change in transcriptional states in ex vivo expanded HSPCs over three sequential weeks (Wk2, Wk3, Wk5). Stacked barplots show the relative proportion of different transcriptional states in the culture over time.

[0017] Figure 2 shows that rapid reconstitution of T lineage shows more clonal stochasticity than other haematopoietic linages by intravenously transplanted HSPCs. (A) A schematic representation of the design of SPLINTR barcoding strategy. (B) A series of FACS contour plots showing fluorescent reporter expression of SPLINTR-barcoded HSPCs.(C) A graphical representation of the percentage of fluorescent reporter gene expression to donor cell blood chimerism (y-axis) three weeks post transplantation with SPLINTR-barcoded HSPCs (x-axis). Each dot represents an individual mouse. (D) A schematic representation of LSK or lineage negative BM cells barcoded with SPLINTR and expanded for either three or five weeks of culture. One third of the culture was transplanted between two 4Gy irradiated C57BL / 6 recipient mice. Erythroid, myeloid, B and T cells were harvested from BM and spleen, or T cell subsets from the thymus at Wk4 post transplantation. (E) A graphical representation of the number of clones (x-axis) in the baseline (TO sample) compared to the 90% percentile engraftment of clones in mice after either 3 or 5 weeks of culture (y-axis). Each dot represents and independent mouse. LSK and Lin. neg. cells were pooled together. (F) A series of graphical representations of barcode contribution (y-axis) to B cells, myeloid (Mye.), T-cell and T-cell progenitors (T-prog.) in the bone marrow (left panel), Spleen (middle panel) or Thymus (right panel). Each dot represents an individual mouse. (G) A graphical representation of barcode distributions in indicated cell lineages derived from barcode-seq from LSK1, LSK2, Lin. neg 1 and Lin. neg 2 pools. Bubble size scales with clone size. Barcodes are ordered in proportion to their frequency in the baseline samples. LSK 2 Lin. neg samples did not pass quality filters for sequencing and are not shown. (H) A graphical representation of the Pearson correlations (y-axis) between replicate mice for individual lineages. Individual cell pools and tissue are indicated (x-axis). (I) A graphical representation of the average percentage contribution of individual clones (bar) in the transplanted LSK1 pool between Wk3 and Wk5 timepoints (y-axis). Venn diagram shows the barcode overlap (pink) between Wk3 (grey) and Wk5 (green)timepoints. Barcode threshold was 90% contribution within an individual mouse. (J) A series of graphical representations of the log2 count per million (CPM) for barcodes in bone marrow lineage samples from mice transplanted with Wk3 (x-axis) and Wk5 (y-axis) culture cells. The CPM was averaged between replicate mice. Each point represents an individual barcode. The R2correlation is displayed.

[0018] Figure 3 shows the engraftment rate and fate of transplanted HSPCs is highly cell intrinsic. (A) A series of FACS contour plots showing silencing of SPLINTR fluorochrome expression in lineage positive and LSK fractions of PVA-cultured HSPCs. (B) A schematic representation of the experimental process to examine silencing of SPLINTR lentivirus in HSPCs grown ex vivo and transplanted into mice. (C) A series of graphical representations of the log2 CPM for barcodes in haematopoietic lineages from mice transplanted with HSPCs cultured for 5w. The CPM was averaged between replicate mice. Each point represents an individual barcode. The R2correlation is displayed. Statistically significant lineage bias is indicated by colours. (D) A graphical representation of the average percentage contribution of individual clones (bar) in the transplanted LSK1 pool between Wk3 and Wk5 timepoints (y-axis). Venn diagram shows the barcode overlap (pink) between Wk3 (grey) and Wk5 (green) timepoints. Barcode threshold was 90% contribution within an individual mouse

[0019] Figure 4 shows that intravenous T cell clonal output is determined by a functional T cell microenvironment. (A) A schematic representation of the experimental process where C57BL / 6 (B6) LSKs were barcoded, expanded and transplanted intravenously into either B6 mice (n = 3) or NSG mice (n = 2). Four weeks later, bone marrow and spleen tissues were harvested and sorted for DNA barcode (BC)-seq of T-cells, B-cells, myeloid, erythroid lineages. scRNA-seq and BC-seq was performed on the baseline TO sample. (B) A graphical representation of the average percentage contribution (y-axis) of individual clones (one clone per bar; x-axis) in all samples harvested from B6 and NSG mice. Venn diagram shows the barcode overlap (green) between BL6 (pink) and NSG (orange) timepoints. Barcode threshold was 95% contribution to an individual mouse. (C) A heatmap representing BC-seq data of individual lineages sorted from B6 or NSG mice. The Top 21 (95thpercentile threshold) clones are grouped by the same proportional output between B6 / NSG recipient mice (green), higher output in B6 recipients (pink) or higher output in NSG recipients (orange). Output is defined by a clone’s proportional output in all samples collected from an individual mouse. This was categorised as high output (>10%,dark purple), medium output (1-10%, purple), low output (<1%, light purple). Statistically significant lineage bias is indicated by star (p<0.05). (D) A series of graphical representations of the log2 CPM for barcodes in lineage samples in B6 (x-axis) and NSG (y-axis) mice. The CPM was averaged between replicate mice. Each point represents an individual barcode. The R2correlation is displayed. (E) A graphical representation of a permutation analysis of Shannon diversity scores derived from the Leiden cluster occupancy (y-axis) of engrafting and non-engrafting cell groups (x-axis). Centre point indicates raw Shannon diversity values with error bars reflecting the standard error of 100 resampling events with replacement. Two-sided Mann Whitney U test. *, p < 2.22 xlO"16. (F) A graphical representation of the top differentially expressed genes in engrafting clones compared versus non-engrafting clones derived from the cells with HSPC Leiden cluster occupancy at the replicate 1 baseline timepoint. Genes of interest are highlighted. Log2 fold change threshold is 0.6, Adjusted p-value < 0.05 Wilcox test. (G) A graphical representation of UMAP projection showing gene set module engraftment score on replicate 1 baseline sample. (H) A graphical representation of the maximum engraftment score (y-axis) for clones with HSPC Leiden cluster occupancy for replicate 1 engrafting (9) and non-engrafting (16), replicate 2, 3 and 4 engrafting (22) and non-engrafting (224) clones (x-axis). (I) A graphical representation of the medium engraftment score (y-axis) for clones with HSPC Louvain cluster occupancy for replicate 1 engrafting (9) and non-engrafting (16), replicate 2, 3 and 4 engrafting (22) and non-engrafting (224) clones (x-axis). Maximum and median score were calculated from all cells within a clone.

[0020] Figure 5 shows that clones with enhanced output in different recipient environments show differential gene expression. (A) A graphical representation of the number of clones (y-axis) in the baseline (TO sample) compared to the 90% percentile engraftment of clones in either C57BL / 6 or NSG mice (x-axis). Each dot represents an independent mouse. (B) A graphical representation of a Principal Component Analysis of DNA barcode -sequencing samples coloured by cell lineage and recipient strain. (C) A graphical representation of UMAP projection showing lymphoid biased clones with the same output (green) or higher in NSG (orange). (D) A graphical representation of the top differentially expressed genes in NSG-hi clones versus same output clones with lymphoid bias. Genes of interest are highlighted. Log2 fold change threshold is 0.6, Adjusted p-value < 0.05 Wilcox test. (E) A graphical representation of UMAP projection showing myeloid-erythroid biased clones with the same output (green) or higher in B6 mice (pink). (F) Agraphical representation of the top differentially expressed genes in B6-hi clones versus same output clones with myeloid / erythroid bias. Genes of interest are highlighted. Log2 fold change threshold is 0.6, Adjusted p-value < 0.05 Wilcox test.

[0021] Figure 6 shows that engrafting clones show enhanced capacity to self-renew in culture. (A) A graphical representation of clonal contribution in vivo (x-axis) compared to frequency (y-axis) in ex vivo expansion. Data is representative of 4 independent experiments.(B) A graphical representation of the number of cells per clone (y-axis) in engrafting and non-engrafting clones (x-axis). (C) A graphical representation of UMAP projections indicating Velocity stream plots and random walk analyses of engrafting and non-engrafting clones in two independent replicates. (D) A graphical representation of UMAP projection showing engrafting cell signature score on published scRNA-seq of ex vivo expanded mouse HSPCs. (E) A series of graphical representations of engraftment score (y-axis) for engrafting and non-engrafting clones (x-axis) with HSPC Louvain cluster occupancy across 4 replicate experiments.

[0022] Figure 7 shows that conditioning of the bone marrow and thymus determines lymphoid output. (A) A schematic representation of experiments where C57BL / 6 LSK cells were barcoded with SPLINTR and expanded for 4 weeks before transplanting the same repertoire of SPLINTR-barcoded sister clones into replicate mice irradiated with either 4Gy or 8Gy TBI (n = 6). Wk4, Wk8 and Wkl6 post transplantation, barcoded T-cells, B-cells, myeloid and erythroid cells were harvested from the BM and spleen for DNA barcode-seq.(B) A graphical representation of the number of clones (y-axis) in the baseline (TO sample) compared to the 95% percentile engraftment of clones in mice at Wk4, Wk8 and Wk 16 (x-axis). Each dot represents an independent mouse. (C) A graphical representation of barcode distributions in indicated cell lineages derived from barcode-seq from Wk4, Wk8 and Wk 16 engraftment in mice irradiated either 4Gy or 8Gy prior to transplantation. Bubble size scales with clone size. Barcodes are ordered in proportion to their frequency in the Wk44Gy mouse 1 myeloid sample. Relative fold output was calculated based on the lineage contribution of barcoded cells in the respective lineage in the spleen. All samples were normalised to the lowest output of a lineage at a given timepoint. (D) A schematic representation of experiments where Lin. neg. HSPCs were barcoded and expanded for four weeks and then injected directly into the thymus of C57BL / 6 mice with either (i) no conditioning (ii) 2.5Gy; (iii) 4.5Gy, (iv) cyclophosphamide, (v) the NSG thymus without conditioning and as a control, (vi) intravenously into 4Gy irradiated mice. (E) A graphical representation of thenumber of clones (y-axis) in the baseline (TO sample) compared to the 90% percentile contribution of clones to T-cells in the spleen four weeks post-transplantation (x-axis). Each dot represents an independent mouse. (F) A heatmap representing BC-seq data of T cells in the spleen from mice that received different conditioning or transplantation sites. The Top 63 clone (95thpercentile threshold) are grouped by conditioning regime with individual mice represent as a column. Relative fold output was calculated based on the tissue chimerism of barcoded T cells in the spleen. All samples were normalised to the lowest output of a lineage (No irradiation ITT).

[0023] Figure 8 shows that intrathymic transplant restricts cell fate to T cell lineage.(A) A series of FACS contour plots showing contribution of barcoded cells transplanted into the tail vein or thymus of syngeneic C57BL / 6 mice. T cell (CD3+), B-cell (CD19+), erythroid (TER-119), myeloid (GR-l+CD 1 lb) in the spleen are indicated. Cells were gated for viability and barcode fluorochrome reporter (VEX-BV510) expression. (B) A graphical representation of T-cell blood chimerism (y-axis) after syngeneic C57BL / 6 transplantation with barcoded HSPCs via the thymus or tail vein (x-axis). (C-D) A series of FACS contour plots showing the (C) thymic selection; and (D) peripheral T cell composition of donor barcoded cells when transplanted directly into the thymus. (E-F) A series of FACS contour plots showing the contribution of barcoded C57BL / 6 donor HSPCs when transplanted directly into the NSG thymus. (G) A photographic representation of C57BL / 6 and NSG harvested thymus Wk4 post intrathymic transplant with C57BL / 6 HSPCs.

[0024] Figure 9 shows that HSPCs engrafted directly in the thymus show normal T-cell differentiation capacity. (A) A schematic representation of the experimental design and analysis. (B) A graphical representation of Pearson correlation (y-axis) comparing barcode contribution to T cell subsets (x-axis) isolated from different peripheral tissues, n = 2. (C) A series of graphical representations of barcode contribution to CD4, CD8 and y8 T-cells. (D) A heatmap showing the top 20 clones ordered by developmental T cell stage- double negative (DN), CD4+CD8+ (DP), CD8+ or CD4+. y8 T-cells are shown separately. (E) A schematic representation of an experiment to address the differentiation of naive T cells isolated after intrathymic or intravenous injection and activated ex vivo. (F) A series of FACS contour plots indicating immunophenotype of cells 5d post bead activation. (G) A series of graphical representations of barcode contribution to naive, central memory (CM) and effector (E) lineages. Bubble size scales with clone size. Barcodes are ordered in proportion to their frequency in the baseline sample. (H) A graphical representation ofPearson correlation (y-axis) of naive, CM and E lineages isolated after intrathymic or intravenous injection (x-axis). (I) A series of FACS contour plots.

[0025] Figure 10 shows that thymic seeding capacity represents the major barrier to rapid T cell reconstitution. (A) A schematic representation of experiments where lineage negative bone marrow was barcoded, expanded for 4 weeks and transplanted the same representation of sister clones into either the tail vein (n = 2), femur (n = 2) or thymus (n = 3). Four weeks later, bone marrow and spleen tissues were harvested and sorted for DNA barcode-seq of T-cells, B-cell, myeloid, erythroid lineages. scRNA-seq and BC-seq was performed on the baseline TO sample. (B) A graphical representation of the number of clones (y-axis) in the baseline (TO sample) compared to the number of clones contributing to T cells in the spleen of mice with HSPCs injected into the vein, into the femur and into the thymus (x-axis). Each dot represents an independent mouse. (C) A heatmap of Pearson correlation comparing all barcode-sequencing samples collected from mice. The heatmap is ordered on transplantation site, cell lineage, tissue of origin and mouse replicates. (D) A series of graphical representations of clones shared in replicate mice by injection site. Overlapping clones between replicate mice are visualised on heat maps ordered by developmental T-cell stage- double negative (DN), CD3- CD4+CD8+ (DP), CD3+DP, SP (CD3+; CD8+ or CD4+), CD3+ (spleen). y8 T-cells are shown separately. (E) A heatmap showing hierarchical clustering of different groups of clones. Clones were classified by fate in vivo-. (Gl) High-output lymphoid clones; (G2) Myeloid / erythroid-biased; (G3) B-cell only; (G4) intrathymic-only; Unbiased. Statistically significant lineage bias is indicated by star <0.05. Statistically significant T-cell differentiation trend is indicated by pink (slow kinetics) or yellow (fast kinetics). (F) A graphical representation of the proportional contribution (y-axis) of clones to donor cells in an indicated lineage.

[0026] Figure 11 shows that engraftment and fate of cells transplanted via the tail vein or femur is similar. (A) A graphical representation of the number of clones (y-axis) in the baseline (TO sample) compared to the number of clones contributing to engraftment with HSPCs injected into the vein, into the femur and into the thymus (x-axis). Each dot represents an independent mouse. (B) A series of graphical representations of the log2 CPM for barcodes in lineage samples from mice transplanted via the tail vein (y-axis) or femur (x-axis). The CPM was averaged between replicate mice. Each point represents an individual barcode. The R2Correlation is displayed. (C) A graphical representation of the temporal lineage bias of clones at Wk4 and Wk8 post transplantation, n = 2 independent experiments.Indicated lineage bias is statistically significant p<0.05. (D) A series of graphical representations of the log2 CPM for barcodes in spleen T cells from mice transplanted with HSPCs via the tail vein, femur or thymus. The CPM was averaged between replicate mice. Each point represents an individual barcode. The R2Correlation is displayed. (E) A graphical representation of a Principal Component Analysis of DNA barcode-sequencing samples coloured by injection site and cell lineage.

[0027] Figure 12 shows that clonal analysis identifies rapid and high output lymphoid program defined by Xlr4b expression. (A) A graphical representation of UMAP projections of baseline scRNA-seq data from Figure 10A. Clones defined in Figure 10E are coloured.(B) A heatmap of hierarchical clustering of individual clone contribution to annotated Leiden clustering in the TO. (C) A series of graphical representations of the top differentially expressed genes in G1 clones versus G2, G3 or G4 clones prior to transplantation (TO sample). G1 versus G2, G1 versus G4 comparisons were derived using cells with occupancy in HSPC Louvain cluster. G1 versus G3 comparison was using all cells in the clones as G3 predominantly was comprised of cells with occupancy in the lymphoid cluster. Genes of interest are highlighted. Log2 fold change threshold is 0.6, adjusted p-value < 0.05 Wilcox test. (D) A graphical representation of logistic regression coefficient for individual gene contribution to G1 clones. (E) A series of FACS contour plots indicating contribution of transgenic donor cells (GFP+) to B-cell lineage in the bone marrow. Cells have been gated on PI for viability and CD45.2+ to identify donor contribution. (F) A graphical representation of contribution (y-axis) of empty vector or Xlr4b over-expressing cells (x-axis) to CD 19+ cells. Each dot represents an individual mouse. Data is normalised to the empty vector control contribution to CD45.1+ donor cells. (G) A graphical representation of contribution (y-axis) of empty vector or Xlr4b over-expressing cells (x-axis) to thymic engraftment. Each dot represents an individual mouse. Data is normalised to the empty vector control contribution to CD45.1+ donor cells, n = 4.

[0028] Figure 13 shows that Xlr4b is heterogeneously expressed in HSPCs. (A) A graphical representation o Xlr4b expression (y-axis) in individual clones (x-axis). Each dot represents an individual cell. (B) A graphical representation of UMAP projection of published ex vivo cultured HSPC data set indicating Xlr4b expression. (C) A graphical representation of UMAP projection ofWk4 mouse lin. neg. BM with cell annotations. (D) A graphical representation of UMAP projection of mouse BM data showing cells with HSC, MPP, CLP, prB and peT / ILC annotation. Expression of key marker genes and Xlr4b aremarked in blue. (E) A graphical representation of UMAP projection showing pseudotime trajectory analysis. (F) A Z-score gene expression heatmap ordered on position of cells along the pseudotime trajectory. Cells annotated as HSC, MPP, CLP are shown.

[0029] Figure 14 shows that overexpression of Xlr4b influences lineage output of transplanted HSPCs. (A) A schematic representation of experiments performed for in vivo assessment o Xlr 4b overexpression. C57BL / 6 (CD45.2+) Lin. neg. BM was transduced with either Xlr4b or empty vector (EV) retrovirus. 4 x 105cells were injected intravenously into C57BL / 5 CD45.1+ recipient mouse irradiated 8Gy. Cells were collected from BM, spleen and thymus at 3 weeks post transplantation, n = 4. (B) A graphical representation of the percentage of fluorochrome expressing donor cells relative to the percentage transduction (y-axis) of HSPCs transduced with either Xlr4b or EV retrovirus (x-axis) ex vivo at transplantation. (C) A graphical representation of donor cell chimerism (y-axis) in the BM and thymus to indicated lineages following transduction with HSPCs transduced with either Xlr4b or EV retrovirus (x-axis). (D) A series of FACS contour plots indicating contribution of transgenic donor cells to GR-1+ myeloid cells in the bone marrow. Cells have been gated on PI for viability and CD45.2+ to identify donor contribution. A graphical representation of contribution (y-axis) of empty vector or Xr 4b over-expressing cells to GR-1+ cells (x-axis). Each dot represents an individual mouse. Data is normalised to the empty vector control contribution to CD45.2+ donor cells. (E) A series of FACS contour plots indicating contribution of transgenic donor cells to T cell progenitor subsets in the thymus.

[0030] Figure 15 shows that Xlr4b is expressed in HSPCs and T cells during embryonic mouse bone development. A graphical representation of the proportion of HSPCs and T cells expressing Xlr4b (y-axis; frequency) versus those lacking detectable expression from embryonic day (E) 16.5, 18.5, postnatal day 0 (P0), and adult stage (x-axis; timepoint). Single-cell RNA-seq data was obtained from Hall et al. (2022, Nature Communications, 13(1): 5403).

[0031] Figure 16 shows that Xlr4b does not influence the immediate homing or expansion of intravenously transplanted HSPCs to the bone marrow and thymus. (A) A graphical representation of the percentage of GFP+ cells in the bone marrow (y-axis; % GFP in BM) and time (y-axis) from C57BL / 6 (CD45.2+) Lin. neg. BM transduced with either Xlr4b (OE) or empty vector (EV) retrovirus and expanded for 5 days. On the day of injection, cell membranes were labelled with CellTace Violet dye (CTV). 5xl05cells were injectedintravenously into C57BL / 6 CD45.1+ recipient mice irradiated 8 Gy. Cells were collected from BM, and thymus at 24 hrs and 72 hrs post transplantation, n = 4. (B) A graphical representation of the percentage of CTV labelling of GFP+ cells (y-axis) and cell division generations (x-axis) in Lin. neg. cells prior to transplantation (0 hrs) and in the bone marrow (24 hrs and 72 hrs). Cell division generations were calculated with the proliferation tool in FlowJo. (C) A graphical representation of the relative number of GFP+ cells (y-axis) in the thymus 24 hrs post injection. 5E5 live cells were analysed. Cell populations were gated on GFP+, CD4-, CD8- Double negative (DN) 1 (CD44+CD25-), DN2 (CD44+CD25+), DN3 (CD44-CD25+) (x-axis). The number of cells quantified was normalized to total number of CD45.1+ host cells. (D) A graphical representation of the relative number of GFP+ cells (y-axis) in the thymus 72 hrs post injection. 5E5 live cells were analysed. Cell populations were gated on GFP+, CD4-, CD8- Double negative (DN) 1 (CD44+CD25-), DN2 (CD44+CD25+), DN3 (CD44-CD25+) (x-axis). The number of cells quantified was normalized to total number of CD45.1+ host cells. (E) A graphical representation of the percentage of B-cells marked by CD 19 expression generated by transplanted GFP+ cells in the BM (y-axis) and time (x-axis; Week 1, Week 2 and Week 12 post transplantation). n=4-6

[0032] Figure 17 shows that Xlr4b regulates bone marrow lymphoid differentiation in common lymphoid progenitor cells (CLPs). (A) A graphical representation of the proportion of annotated cell types following label transfer-based annotation of single-cell RNA sequencing (scRNA-seq) data from mouse Lin. neg. bone marrow cells collected 1 week after transplantation with HSPCs overexpressing Xlr4b (OE) or an empty vector (EV) using the dataset from Pei etal. (2020, Cell Stem Cell, 27(3): 383-395). n = 2 mice per timepoint.(B) A graphical representation of the number of B cells in the mouse samples used for scRNA-seq in (A) Data indicates the percentage of B-cells marked by CD 19 expression in transplanted GFP+ cells in the bone marrow (y-axis) at Wkl . n = 2 mice per timepoint. (C) A graphical representation of the number of GFP+ cells (y-axis) in the thymus 72 hrs post injection. 5E5 live cells were analysed. Cell populations were gated on GFP+, CD4-, CD8-Double negative (DN) 1 (CD44+CD25-), DN2 (CD44+CD25+), DN3 (CD44-CD25+). The number of cells quantified was normalized to total number of CD45.1+ host cells, n = 2 mice per timepoint. (D) A graphical representation of the proportion of each annotated cell type based on label transfer from Pei etal. (2020, supra). A consistent reduction in pro-B (prB) cells is observed in the Xlr4b OE condition, n = 2 mice per timepoint. (E) A heatmapshowing relative activity of key transcription factor regulons involved in B-cell development analysed by SCENIC differential regulon analysis of scRNA-seq data.

[0033] Figure 18 shows that ectopic expression of FAM9B regulates bone marrow lymphoid differentiation. A graphical representation of the contribution (y-axis; VEX% contribution) of lentiviral empty vector (left panel; EV) or FAM9B over-expressing (right panel; OE) cells to B-cells and thymic engraftment within each mouse. Each dot represents an individual mouse. Dotted line indicates 0. The mean of the differences between B-cell and thymic contribution is indicated by the line. Lentiviral expressing cells are marked by VEX fluorescent protein expression, n = 3.Brief Description of the Sequences

[0034] Nucleic acid and amino acid sequences are referred to by sequence identifier (SEQ ID), with reference to the accompanying sequence listing.

[0035] SEQ ID NO: 1 is a nucleotide sequence showing the coding sequence (CDS) of Xlr4a. transcript 1.

[0036] SEQ ID NO: 2 is a nucleotide sequence showing the CDS of Xlr4a. transcript 2.

[0037] SEQ ID NO: 3 is a nucleotide sequence showing the CDS of Xlr4a. transcript 3.

[0038] SEQ ID NO: 4 is a nucleotide sequence showing the CDS of Xlr4b. transcript 1.

[0039] SEQ ID NO: 5 is a nucleotide sequence showing the CDS of Xlr4b. transcript 2.

[0040] SEQ ID NO: 6 is a nucleotide sequence showing the CDS of Xlr4b. transcript 3.

[0041] SEQ ID NO: 7 is a nucleotide sequence showing the CDS of Xlr4b. transcript 4.

[0042] SEQ ID NO: 8 is a nucleotide sequence showing the CDS of Xlr4b. transcript 8.

[0043] SEQ ID NO: 9 is a nucleotide sequence showing the CDS of FAM9A, transcript 1.

[0044] SEQ ID NO: 10 is a nucleotide sequence showing the CDS of FAM9A, transcript 2.

[0045] SEQ ID NO: 11 is a nucleotide sequence showing the CDS of FAM9B, transcript 1.

[0046] SEQ ID NO: 12 is a nucleotide sequence showing the CDS of FAM9B, transcript 2.

[0047] SEQ ID NO: 13 is a nucleotide sequence showing the CDS of FAM9B, transcript 3.

[0048] SEQ ID NO: 14 is a nucleotide sequence showing the CDS of FAM9B, transcript 5.

[0049] SEQ ID NO: 15 is a nucleotide sequence showing the CDS of FAM9B, transcript 6.

[0050] SEQ ID NO: 16 is a nucleotide sequence showing the CDS of FAM9C, transcript 1.

[0051] SEQ ID NO: 17 is a nucleotide sequence showing the CDS of FAM9C, transcript 2.

[0052] SEQ ID NO: 18 is a nucleotide sequence showing the CDS of FAM9C, transcript 3.

[0053] SEQ ID NO: 19 is a nucleotide sequence showing the CDS of FAM9C, transcript 4.

[0054] SEQ ID NO: 20 is a nucleotide sequence showing the CDS of FAM9C, transcript 5.

[0055] SEQ ID NO: 21 is a codon-optimised nucleotide sequence encoding Xlr4b.

[0056] SEQ ID NO: 22 is the nucleotide sequence of a nucleic acid construct encoding Xlr4b

[0057] SEQ ID NO: 23 is the nucleotide sequence of a vector comprising the nucleic acid construct of SEQ ID NO: 21.

[0058] SEQ ID NO: 24 is a codon-optimised nucleotide sequence encoding FAM9B (corresponding to the CDS of SEQ ID NO: 11).

[0059] SEQ ID NO: 25 is the nucleotide sequence of a vector comprising the codon-optimised nucleotide sequence of SEQ ID NO: 24.Detailed Description

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0061] All sequence database identifiers (e.g., GenBank ID, EMBL-Bank ID, DNA Data Bank of Japan (DDBJ) ID, etc.) provided herein were current at the filing date.

[0062] The articles "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells (e.g., a population of cells); reference to "an ortholog" includes a single ortholog, as well as two or more orthologs; and so forth.

[0063] In the context of this specification, the term “about” is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 40%-60%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”.

[0064] Throughout this specification and the claims that follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0065] The term “optionally” is used herein to mean that the subsequent described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiment in which the event or circumstance occurs as well as embodiments in which it does not.

[0066] As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.Engineered haematopoietic or progenitor stem cells

[0067] In an aspect disclosed herein, there is provided an engineered haematopoietic stem or progenitor cell (HSPC), which has been modified to ectopically express an X-linkedlymphocyte-regulated (Xlr) gene or an ortholog thereof relative to an equivalent unmodified HSPC.

[0068] “Haematopoietic stem or progenitor cells” or “HSPC” are CD34+ stem cells capable of giving rise to both mature myeloid and lymphoid cell types, including T lymphocytes, natural killer (NK) cells, and B lymphocytes. Suitable HSPCs would be known to persons skilled in the art, illustrative examples of which include hematopoietic stem cells (HSCs), long-term haematopoietic stem cells (LT-HSCs), short-term haematopoietic stem cells (ST-HSCs), and multipotent progenitor cells (MPPs).

[0069] In an embodiment, the HSPC is an HSC or an MPP.

[0070] In an embodiment, the HSPC is an HSC.

[0071] HSPC are obtainable from suitable sources that would be known to persons skilled in the art, illustrative embodiments of which include bone marrow, umbilical cord blood, cord blood-derived stem cells, mesenchymal stem cells, haematopoietic stem cells differentiated from induced pluripotent stem cells, and peripheral blood.

[0072] The HSPC contemplated herein may be derived from any species, particularly a vertebrate, and even more particularly a mammal. Suitable vertebrates that fall within the scope of the disclosure include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatto)) and baboon (Papio ur sinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In a preferred embodiment, the cells are derived from a human.

[0073] The terms “X -linked lymphocyte-regulated gene” and "Xlr gene” are used interchangeably herein to refer to a group of genes on the X chromosome, which encodeproteins having a CORI domain. Suitable Xlr genes an orthologs thereof would be known to persons skilled in the art, illustrative examples of which include Six, Xmr, Slxll, Sly, Sycp3, Xlr3 (i.e., Xr3a, Xlr3b, Xlr3c), Xlr4 (i.e., Xlr4a, Xlr4b, Xlr4c), Xlr5 (i.e., Xlr5a, Xlr5b, Xr5c), FAM9A, FAM9B, and FAM9C.

[0074] In an embodiment, the Xlr gene is selected from the group consisting of Xlr 4a and Xlr 4b. In another embodiment, the Xr gene is Xlr 4b.

[0075] The term “ortholog” as used herein refers to homologous genes or proteins that are descendant from a common ancestor but separated by speciation.

[0076] In an embodiment, the ortholog is a human ortholog of an Xr gene.

[0077] In an embodiment, the ortholog is selected from the group consisting of FAM9A, FAM9B and FAM9C.

[0078] In an embodiment, the ortholog is FAM9A.

[0079] In an embodiment, the ortholog is FAM9B.

[0080] In an embodiment, the ortholog is FAM9C.

[0081] The term “ectopic expression” as used herein refers to the abnormal expression of a gene (or its resulting protein) in a cell (e.g., overexpression, expression at a different developmental stage, expression in a different cell type, etc.).

[0082] Methods for ectopically expressing a gene of interest would be known to persons skilled in the art, illustrative examples of which include introduction of exogenous nucleic acid constructs by transfection, electroporation, nucleofection, transduction (e.g., viral and non-viral transduction), nanoparticle-based gene delivery, and gene editing (e.g., using meganucleases, zinc finger nucleases (ZFN), clustered regularly interspaced short palindromic repeats (CRISPR), and transcription activator-like effector nuclease (TALENs)).

[0083] In an embodiment, the exogenous nucleic acid construct has been introduced by gene editing, transfection or transduction.

[0084] In an embodiment, the modification includes increased expression of an Xlr gene or an ortholog thereof relative to an unmodified equivalent HSPC.

[0085] In an embodiment, expression of an Xlr gene or an ortholog thereof is increased by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) relative to an unmodified equivalent HSC.

[0086] In an embodiment, the engineered HSPC comprises an exogenous nucleic acid construct encoding an XLR protein or an ortholog thereof.

[0087] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0088] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0089] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0090] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 1-20.

[0091] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 9-20, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%.89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0092] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 9-20, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0093] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 9-20, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0094] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 9-20.

[0095] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 11-15, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%.89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0096] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 11-15, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0097] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 11-15, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0098] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 11-15.

[0099] In an embodiment, the exogenous nucleic acid construct comprises a sequence of SEQ ID NO: 11, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0100] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence of SEQ ID NO: 11, or a sequence having at least 75% sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0101] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence of SEQ ID NO: 11, or a sequence having at least 78% sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0102] In an embodiment, the exogenous nucleic acid construct comprises a sequence of SEQ ID NO: 11.

[0103] In an embodiment, there is provided an engineered HSPC, which has been modified to ectopically express Xlr4a relative to an equivalent unmodified HSPC.

[0104] In an embodiment, there is provided an engineered HSPC, which has been modified to ectopically express Xlr4b relative to an equivalent unmodified HSPC.

[0015] In an embodiment, there is provided an engineered HSPC, which has been modified to ectopically express FAM9A relative to an equivalent unmodified HSPC.

[0106] In an embodiment, there is provided an engineered HSPC, which has been modified to ectopically express FAM9B relative to an equivalent unmodified HSPC.

[0107] In an embodiment, there is provided an engineered HSPC, which has been modified to ectopically express FAM9C relative to an equivalent unmodified HSPC.

[0108] In another aspect disclosed herein, there is provided an engineered lymphoid cell, which has been modified to ectopically express an Xr gene or an ortholog thereof relative to an equivalent unmodified lymphoid cell.100109] In an embodiment the lymphoid cell is selected from the group consisting of a lymphoid progenitor cell, an innate lymphoid cell, a T lymphocyte, and a natural killer (NK) cell.

[0110] In an embodiment, the lymphoid cell is a T lymphocyte.

[0111] Suitable T lymphocytes would be known to persons skilled in the art, illustrative examples of which include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, activated T lymphocytes or tumour infiltrating lymphocytes (TILs). Illustrative populations of T lymphocytes suitable for use in particular embodiments include but are not limited to helper T lymphocytes (CD4+T cell), cytotoxic T lymphocytes (CD8+T cell), CD4+CD8+T cell, CD4 CD8" T cell, or any other subset of T lymphocytes. Other illustrative populations of T lymphocytes suitable for use in particular embodiments include but are not limited to T lymphocytes expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L,CD 127, CD 197, and HLA-DR and if desired, can be further isolated by positive or negative selection techniques.

[0112] In an embodiment, the modification includes increased expression of an Xlr gene or an ortholog thereof relative to an unmodified equivalent lymphoid cell.

[0113] In an embodiment, expression of an Xlr gene or an ortholog thereof is increased by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) relative to an unmodified equivalent lymphoid cell.

[0114] In an embodiment, the engineered lymphoid cell comprises an exogenous nucleic acid construct encoding an XLR protein or an ortholog thereof.

[0115] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%.85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0116] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0117] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0118] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 1-20.

[0119] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 9-20, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%.89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0120] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 9-20, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0121] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 9-20, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0122] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 9-20.

[0123] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 11-15, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%.89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0124] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 11-15, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0125] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 11-15, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0126] In an embodiment, the exogenous nucleic acid construct comprises a sequence selected from SEQ ID NOs: 11-15.

[0127] In an embodiment, the exogenous nucleic acid construct comprises a sequence of SEQ ID NO: 11, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0128] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence of SEQ ID NO: 11, or a sequence having at least 75% sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0129] In an embodiment, the exogenous nucleic acid construct comprises a nucleotide sequence of SEQ ID NO: 11, or a sequence having at least 78% sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0130] In an embodiment, the exogenous nucleic acid construct comprises a sequence of SEQ ID NO: 11.

[0131] In an embodiment, there is provided an engineered lymphoid cell, which has been modified to ectopically express Xlr4a relative to an equivalent unmodified lymphoid cell.

[0132] In an embodiment, there is provided an engineered lymphoid cell, which has been modified to ectopically express Xlr4b relative to an equivalent unmodified lymphoid cell.

[0133] In an embodiment, there is provided an engineered lymphoid cell, which has been modified to ectopically express FAM9A relative to an equivalent unmodified lymphoid cell.

[0134] In an embodiment, there is provided an engineered lymphoid cell, which has been modified to ectopically express FAM9B relative to an equivalent unmodified lymphoid cell.

[0135] In an embodiment, there is provided an engineered lymphoid cell, which has been modified to ectopically express FAM9C relative to an equivalent unmodified lymphoid cell.Compositions

[0136] In another aspect disclosed herein, there is provided a composition comprising a population of the engineered HSPC disclosed elsewhere herein.

[0137] In another aspect disclosed herein, there is provided a composition comprising a population of the engineered lymphoid cell disclosed elsewhere herein.

[0138] The term "composition" as used herein refers to a composition that is in a form that allows the biological activity of the active ingredient (i.e., the engineered HSPC or lymphoid cell disclosed herein) to be effective, and that does not contain additionalingredients that have unacceptable toxicity to the subject to which the composition is to be administered.

[0139] In an embodiment, the composition comprises a population of the engineered HSPC or lymphoid cell disclosed herein in sufficient number to administer a dosage of 104to 109cells / kg body weight. Accordingly, the composition may comprise a population of the engineered HSC or lymphoid cell disclosed herein in sufficient number to administer a dosage of 104, 105, 106, 107, 108or 109cells / kg body weight.

[0140] In an embodiment, the composition comprises a population of the engineered HSPC or lymphoid cell disclosed herein in sufficient number to administer a dosage of 105to 106cells / kg body weight.

[0141] In an embodiment, the composition is suitable for intravenous administration.

[0142] The composition disclosed herein may be prepared according to conventional methods well known in the pharmaceutical industries, such as those described in Remington’s Pharmaceutical Handbook (Mack Publishing Co., NY, USA), comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.

[0143] In an embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0144] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain anti-oxidants, buffers and solutes to render the composition isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, anti-fungal and anti-bacterial agents, isotonic and absorption agents, and the like.

[0145] It is further contemplated herein that the composition may be co-administered with one or more other agents suitable for the treatment of, or amelioration of symptoms associated with, a haematological disease, illustrative examples of which include surgery, chemotherapy (e.g., anastrozole, bicalutamide, bleomycin sulfate, busulfan, busulfan injection, capecitabine, N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosinearabinoside, cytarabine liposome injection, dacarbazine, dactinomycin, daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, fludarabine phosphate, 5- fluorouracil, flutamide, tezacitibine, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, L-asparaginase, leucovorin calcium, melphalan, 6-mercaptopurine, methotrexate, mitoxantrone, mylotarg, paclitaxel, phoenix (Yttrium90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant, tamoxifen citrate, teniposide, 6-thioguanine, thiotepa, tirapazamine, topotecan hydrochloride for injection, vinblastine, vincristine, and vinorelbine), radiation, immunosuppressive agents (e.g., cyclosporin, azathioprine, methotrexate, my cophenolate, and FK506), antibodies, or other immunoablative agents (e.g., CAMPATH), targeted agents, steroids, and peptide vaccines.

[0146] Such combinations may be administered before, simultaneous with, concurrent with or after the composition.Nucleic acid constructs and vectors

[0147] In another aspect disclosed herein, there is provided a nucleic acid construct comprising a nucleotide sequence encoding an XLR protein or an ortholog thereof.

[0148] As used herein the terms “polynucleotide”, “nucleic acid” or “nucleic acid molecule” mean a single- or double-stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues or natural nucleotides, or mixtures thereof, and can include molecules comprising coding and non-coding sequences of a gene, sense and antisense sequences and complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.

[0149] The term “nucleotide” as used herein refers to the nucleotides adenosine, guanosine, cytidine, thymidine and uridine, each of which comprise a nucleotide base attached to a ribose ring. A person skilled in the art will appreciate that the terms "adenine / adenosine", "uracil / uridine", "guanine / guanosine", "cytosine / cytidine" and "thymidine / thymine" may be used interchangeably herein with the single letters A, U, G, C and T, respectively, which refer the nucleotide base comprised by the nucleotides.

[0150] The terms "non-naturally occurring", "engineered" or "recombinant" may be interchangeably used herein to refer to nucleotide sequences or nucleic acid molecules that are distinguished from their naturally occurring counterparts. For example, the nucleic acid construct of the present disclosure may be recombinant, synthetic, or comprise mixtures of naturally and non-naturally occurring nucleotides. Non-naturally occurring nucleotides or nucleotide analogs may be modified at the ribose, phosphate and / or base moiety.

[0151] As used herein, the terms “encode”, “encoding” and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid is said to "encode" a polypeptide if it can be transcribed and / or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and / or translated to produce the polypeptide. Such a nucleic acid may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode," "encoding" and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.100152] Suitable XLR proteins and orthologs thereof would be known to persons skilled in the art, illustrative examples of which include SLX / XMR, SLXL1, SLY, SYCP3, XLR3 (i.e., XLR3A, XLR3B, XLR3C), XLR4 (i.e., XLR4A, XLR4B, XLR4C), XLR5 (i.e., XLR5A, XLR5B, XLR5C), FAM9A, FAM9B, and FAM9C.

[0153] In an embodiment, the XLR protein is selected from the group consisting of XLR4A and XLR4B. In another embodiment, the XLR protein is XLR4B.

[0154] In an embodiment, the ortholog is a human ortholog of an XLR protein.

[0155] In an embodiment, the ortholog is selected from the group consisting of FAM9A, FAM9B and FAM9C.

[0156] In an embodiment, the ortholog is FAM9A.

[0157] In an embodiment, the ortholog is FAM9B.

[0158] In an embodiment, the ortholog is FAM9C.

[0159] In an embodiment, the nucleic acid construct is a codon optimised nucleic acid sequence for expression in particular cells, e.g., SEQ ID NOs: 21 and 24. In general, codon optimisation refers to a process of modifying a nucleotide sequence for enhanced expression in the cell of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (i.e. , differences in codon usage between organisms) often correlates with the efficiency of translation of mRNA, which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.

[0160] The nucleic acid construct of the present disclosure may be produced using any method in the art, including synthetically or by recombinant techniques such as expression of polynucleotide constructs encoding the components.

[0161] In an embodiment, the nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0162] Methods for the determination of nucleotide sequence identity would be known to persons skilled in the art, illustrative examples of which include computer programs that employ algorithms such as protein BLAST (Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402).

[0163] In an embodiment, the nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0164] In an embodiment, the nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

[0165] In an embodiment, the nucleic acid construct comprises a sequence selected from SEQ ID NOs: 1-20.

[0166] In an embodiment, the nucleic acid construct comprises a sequence selected from SEQ ID NOs: 9-20, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0167] In an embodiment, the nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 9-20, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0168] In an embodiment, the nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 9-20, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-20.

[0169] In an embodiment, the nucleic acid construct comprises a sequence selected from SEQ ID NOs: 9-20.

[0170] In an embodiment, the nucleic acid construct comprises a sequence selected from SEQ ID NOs: 11-15, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0171] In an embodiment, the nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 11-15, or a sequence having at least 75% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0172] In an embodiment, the nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 11-15, or a sequence having at least 78% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-15.

[0173] In an embodiment, the nucleic acid construct comprises a sequence selected from SEQ ID NOs: 11-15.

[0174] In an embodiment, the nucleic acid construct comprises a sequence of SEQ ID NO: 11, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0175] In an embodiment, the nucleic acid construct comprises a nucleotide sequence of SEQ ID NO: 11, or a sequence having at least 75% sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0176] In an embodiment, the nucleic acid construct comprises a nucleotide sequence of SEQ ID NO: 11, or a sequence having at least 78% sequence identity to a nucleotide sequence of SEQ ID NO: 11.

[0177] In an embodiment, the nucleic acid construct comprises a sequence of SEQ ID NO: 11.

[0178] In an embodiment, the nucleic acid construct further comprises a promoter.

[0179] The term "promoter" as used herein refers to an array of nucleic acid control sequences that direct the transcription of the nucleotide sequence comprises in the nucleic acid construct. Suitable promoters would be known to persons skilled in the art, illustrative examples of which include retroviral LTR elements, constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, or [3-actin, inducible promoters, such as those containing Tet-operator elements, and / or tissue specific promoters.

[0180] In an embodiment, the nucleic acid construct further comprises other additional regulatory elements or sequences.

[0181] Suitable regulatory sequences would be known to persons skilled in the art, illustrative examples of which include leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, and enhancer or activator sequences.

[0182] In another aspect disclosed herein, there is provided a vector comprising the nucleic acid construct disclosed elsewhere herein.

[0183] The vectors can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into a host cell genome. Vectors may be replication competent or replication-deficient. Exemplary vectors include, but are not limited to, plasmids, cosmids, and viral vectors, such as adeno-associated virus (AAV) vectors, lentiviral, retroviral, adenoviral, herpesviral, parvoviral and hepatitis viral vectors. The choice and design of an appropriate vector is within the ability and discretion of the persons skilled in the art. Preferably, however, the vector is suitable for use in biotechnology.

[0184] Vectors suitable for use in biotechnology would be known to persons skilled in the art, illustrative examples of which include viral vectors derived from adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, self-amplifying single-strand RNA (ssRNA) viruses such as alphavirus (e.g., Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis, Ml), and flavivirus (e.g., Kunjin virus, West Nile virus, Dengue virus), rhabdovirus (e.g, rabies, vesicular stomatitis virus), measles virus, Newcastle Disease virus (NDV) and poxivirus as described by, for example, Lundstrom (2019, Diseases, 6: 42).

[0185] In an embodiment, the vector is a plasmid or a viral vector.

[0186] In an embodiment, the vector comprises a nucleotide sequence of SEQ ID NOs: 23 or 25, or a sequence having at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a nucleotide sequence of SEQ ID NOs: 23 or 25.

[0187] In an embodiment, the vector comprises, consists, or consists essentially of a nucleotide sequence of SEQ ID NOs: 23 or 25.

[0188] The present disclosure also provides non-viral delivery vehicles of the nucleic acid construct and vectors disclosed herein, and components thereof. Suitable non-viraldelivery vehicles will be known to persons skilled in the art, illustrative examples of which include using lipids, lipid-like materials or polymeric materials, as described by, e.g., Rui et al. (2019, Trends in Biotechnology, 37(3): 281-293), and nanoparticles / nanocarriers, as described by, e.g., Nguyen et al. (2020, Nature Biotechnology, 38: 44-49).

[0189] The HSPC may be provided with the nucleic acid molecules or vectors described herein using any suitable method known in the art. Such methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like. The skilled person would readily understand and adapt any such method taking consideration of whether the nucleic acid construct is provided as a polynucleotide or vector.Methods for enhancing T cell reconstitution following haematopoietic stem cell transplantation

[0190] In another aspect disclosed herein, there is provided a method for enhancing T cell reconstitution following haematopoietic stem cell transplantation, the method comprising administering an effective amount of the engineered HSPC (e.g., a population of engineered HSPCs) or the composition disclosed elsewhere herein to a subject in need thereof.

[0191] In an embodiment, the endogenous haematopoietic compartment of the subject has been depleted prior to the administration of the engineered HSPC (e.g., a population of engineered HSPCs) or the composition.

[0192] The terms “deplete”, "depleting", “depleted” and the like are used interchangeably herein to mean reducing the endogenous haematopoietic compartment in a subject in preparation for haematopoietic stem cell transplantation. It is to be understood that the terms “deplete”, "depleting", “depleted” and the like, as used herein, do not imply that the entire endogenous haematopoietic compartment has been eliminated or is no longer evident. Said depletion may also reduce the endogenous haematopoietic compartment, e.g., by the selective reduction of subpopulations of the endogenous haematopoietic compartment, non-myeloablative or reduced intensity conditioning regimens.

[0193] Methods for the depletion of the endogenous haematopoietic compartment would be known to persons skilled in the art, illustrative examples of which include theadministration of conditioning therapies, e.g., chemotherapy, radiotherapy, immunotherapy and combinations of the foregoing.

[0194] In an embodiment, the endogenous haematopoietic compartment has been depleted by chemotherapy, radiotherapy, immunotherapy, or combinations of the foregoing.

[0195] In an embodiment, the subject has a haematological disease.

[0196] In an embodiment, the haematological disease is selected from the group consisting of multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelofibrosis, essential thrombocytosis, polycythemia vera, solid tumors, aplastic anemia, severe combined immune deficiency syndrome (SCID), thalassemia, sickle cell disease, chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anaemia, Blackfan-Diamond anaemia, enzymatic disorders, non-malignant autoimmune diseases, neuromyelitis optica and multiple sclerosis.

[0197] In an embodiment, the haematological disease is a malignant disease.

[0198] In an embodiment, the malignant disease is selected from the group consisting of multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelofibrosis, essential thrombocytosis, polycythemia vera, solid tumors (e.g., testicular germ cell tumors, medulloblastoma, and breast cancer).

[0199] In an embodiment, the haematological disease is a non-malignant disease.100200] In an embodiment, the non-malignant disease is selected from the group consisting of aplastic anemia, severe combined immune deficiency syndrome (SCID), thalassemia, sickle cell disease, chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anaemia, Blackfan-Diamond anaemia, enzymatic disorders, non-malignant autoimmune diseases (e.g., systemic sclerosis and systemic lupus erythematosus), neuromyelitis optica and multiple sclerosis.

[0201] The term “subject” as used herein refers to any mammal, including livestock and other farm animals (such as cattle, goats, sheep, horses, pigs and chickens), performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals and humans. In an embodiment, the subject is a human. In an embodiment, the subject is an adult. In another embodiment, the subject is a child.

[0202] In an embodiment, the engineered HSPC (e.g., the population of engineered HSPCs) are autologous to the subject.

[0203] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later to be administered into the subject in accordance with the methods disclosed herein. Accordingly, in certain embodiments, HSC isolated from the subject may be contacted with the nucleic acid construct described herein and cultured in vitro or ex vivo for a time and under conditions suitable for the integration of the nucleic acid construct, before being reinfused back into the subject in accordance with the method of treatment disclosed elsewhere herein.

[0204] In an embodiment, the engineered HSPC (e.g., a population of engineered HSPCs) are allogenic to the subject.

[0205] The term "allogenic" as used herein refers to any material derived from a different animal of the same species (i.e., a donor) as the subject to whom the material is administered.

[0206] In an embodiment, the allogenic donor is HLA-matched to the subj ect. In another embodiment, the allogenic donor is haploidentical to the subject.

[0207] In another aspect, there is provided a method for enhancing T cell reconstitution following haematopoietic stem cell transplantation, the method comprising administering an effective amount of an HSPC (e.g., a population of HSPCs) to a subject in need thereof, wherein the HSPC is characterised by (i) multilineage potential; and (ii) lymphoid bias, and wherein characteristics (i) and (ii) are independent of the route of administration of the HSPC.

[0208] In an embodiment, the hematopoietic stem cell transplantation comprises administration of the engineered HSPC (e.g., a population of engineered HSPCs) disclosed herein.Methods for selecting HSPCs that are capable of enhanced T cell reconstitution

[0209] In another aspect, there is provided a method for selecting HSPC (e.g., a population of HSPC) that is capable of enhanced T cell reconstitution following haematopoietic stem cell transplant, the method comprising:(a) obtaining a plurality of HSPCs;(b) generating single cell expression data for each of the plurality of HSPC; (c) based on the expression data generated in step (b), determining the level of expression of an Xlr gene or an ortholog thereof in each of the plurality of HSPC; and(d) selecting HSPC, or a population thereof, with differential expression of an Xlr gene or an ortholog thereof relative to a reference level.

[0210] In an embodiment, the Xlr genes an orthologs thereof are selected from the group consisting of Six, Xmr, Slxll, Sly, Sycp3, Xlr 3 (i.e., Xlr 3a, Xlr 3b, Xlr 3c), Xlr 4 (i.e., Xlr 4a, Xlr4b,Xlr4c),Xlr5 (i.e., Xlr5a, Xlr 5b, Xlr5c). FAM9A, FAM9B, FAM9C, and combinations of the foregoing.

[0211] In an embodiment, the Xlr gene is selected from the group consisting of Xlr 4a and Xlr 4b. In another embodiment, the Mr gene is Xlr 4b.

[0212] The term “ortholog” as used herein refers to homologous genes or proteins that are descendant from a common ancestor but separated by speciation.

[0213] In an embodiment, the ortholog is a human ortholog of an Mr gene.

[0214] In an embodiment, the ortholog is selected from the group consisting of FAM9A, FAM9B and FAM9C.

[0215] In an embodiment, the ortholog is FAM9A.

[0216] In an embodiment, the ortholog is FAM9B.

[0217] In an embodiment, the ortholog is FAM9C.

[0218] In an embodiment, the reference level is a known or predetermined level of expression of an Xlr gene or an ortholog thereof that is representative of the level of expression of an Xlr gene or an ortholog thereof in an unmodified (e.g., wild-type) HSPC. In another embodiment, the comparison may be made to the expression of an Xlr gene or an ortholog thereof by an unmodified HSPC at a particular development stage. Alternatively, the comparison may be made to the level of expression of an Xlr gene or an ortholog thereof in a different single HSPC from the plurality of HSPCs obtained in step (a).

[0219] In an embodiment, the expression data is generated by single cell sequencing.

[0220] In an embodiment, the method further comprises expanding the HSPC, or a population thereof, selected in step (d) to provide an expanded population of HSPCs.

[0221] In an embodiment, the method further comprising using the selected HSPC, or a population thereof, or the expanded population of HSPCs for therapy (e.g., haematopoietic stem cell transplantation).

[0222] In an embodiment, the method further comprises culturing the expanded population of HSPCs for a time and under conditions to induce T cell or T cell-like differentiation.

[0223] In an embodiment, the method further comprises determining the level of expression of one or more o Flt3, Satbl,Il7r, Dnii. or orthologs of any one of the foregoing.Methods for the treatment of a haematological disease and associated therapeutic uses

[0224] In another aspect disclosed herein, there is provided a method for the treatment of a haematological disease, the method comprising administering the engineered HSPC (e.g., a population of engineered HSPCs), or the composition disclosed elsewhere herein to a subject in need thereof.

[0225] In another aspect disclosed herein, there is provided a use of the engineered HSPC (e.g., a population of engineered HSPCs), or the composition disclosed herein in the manufacture of a medicament for the treatment of a haematological disease.

[0226] In another aspect disclosed herein, there is provided a method for the treatment of a haematological disease, the method comprising administering the engineered lymphoidcell (e.g., a population of engineered lymphoid cells), or the composition disclosed elsewhere herein to a subject in need thereof.

[0227] In another aspect disclosed herein, there is provided a use of the engineered lymphoid cell (e.g., apopulation of engineered lymphoid cells), orthe composition disclosed herein in the manufacture of a medicament for the treatment of a haematological disease.

[0228] In an embodiment, the haematological disease is selected from the group consisting of multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelofibrosis, essential thrombocytosis, polycythemia vera, solid tumors, aplastic anemia, severe combined immune deficiency syndrome (SCID), thalassemia, sickle cell disease, chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anaemia, Blackfan-Diamond anaemia, enzymatic disorders, non-malignant autoimmune diseases, neuromyelitis optica and multiple sclerosis.

[0229] In an embodiment, the haematological disease is a malignant disease.

[0230] In an embodiment, the malignant disease is selected from the group consisting of multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelofibrosis, essential thrombocytosis, polycythemia vera, solid tumors (e.g., testicular germ cell tumors, medulloblastoma, and breast cancer).

[0231] In an embodiment, the haematological disease is a non-malignant disease.

[0232] In an embodiment, the non-malignant disease is selected from the group consisting of aplastic anemia, severe combined immune deficiency syndrome (SCID), thalassemia, sickle cell disease, chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anaemia, Blackfan-Diamond anaemia, enzymatic disorders, non-malignant autoimmune diseases (e.g., systemic sclerosis and systemic lupus erythematosus), neuromyelitis optica and multiple sclerosis.

[0233] The therapeutic regimen for the treatment of haematological diseases can be determined by a person skilled in the art and will typically depend on factors including, butnot limited to, the type and stage of the haematological disease in addition to the age, weight and general health of the subject. Another determinative factor may be the risk of developing recurrent disease. For instance, for a subject identified as being at high risk or higher risk or developing recurrent disease, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is deemed at a low or lower risk of developing recurrent disease.

[0234] As used herein, the term “effective amount” typically refers to an amount of the engineered HSPC (e.g., a population of engineered HSPCs) or engineered lymphoid cell (e.g., a population of engineered lymphoid cells), or compositions disclosed herein that is sufficient to affect one or more beneficial or desired therapeutic outcomes (e.g., treatment of, or amelioration of the symptoms of the haematological disease). Said beneficial or desired therapeutic outcomes may be measured using clinical techniques known in the art, illustrative examples of which include the measurement of imaging biomarkers, tumor size (e.g., as measured by anatomical imaging modalities, such as CT or MRI), quantification of the presence of inflammatory mediators (e.g., Interleukin- 1, TNF, TGF-[3, etc.). An “effective amount” can be provided in one or more administrations.

[0235] The terms “treat”, "treating", “treatment” and the like are used interchangeably herein to mean relieving, reducing, alleviating, ameliorating or otherwise inhibiting the severity and / or progression of cancer, or a symptom thereof, in a subject. It is to be understood that the terms “treat”, "treating", “treatment” and the like, as used herein, do not imply that a subject is treated until clinical symptoms of the haematological disease have been eliminated or are no longer evident. Said treatment may also reduce the severity of the haematological disease by preventing progression or alleviating the symptoms associated with the haematological disease.|00236] In an embodiment, the engineered HSPC (e.g., a population of engineered HSPCs) or engineered lymphoid cells (e.g., a population of engineered lymphoid cells) are autologous to the subject.

[0237] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later to be administered into the subject in accordance with the methods disclosed herein. Accordingly, in certain embodiments, HSC isolated from the subject may be contacted with the nucleic acid construct described herein and cultured in vitro or ex vivo for a time and under conditions suitable for the integration of the nucleic acidconstruct, before being reinfused back into the subject in accordance with the method of treatment disclosed elsewhere herein.

[0238] In an embodiment, the engineered HSPC (e.g., a population of engineered HSPCs) or engineered lymphoid cells (e.g., a population of engineered lymphoid cells) are allogenic to the subject.

[0239] The term "allogenic" as used herein refers to any material derived from a different animal of the same species (i.e., a donor) as the subject to whom the material is administered.

[0240] In an embodiment, the allogenic donor is HLA-matched to the subj ect. In another embodiment, the allogenic donor is haploidentical to the subject.

[0241] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the present disclosure without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0242] The present disclosure will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the disclosure.Example 1Materials and methodsMouse details and strain

[0243] All animal experiments were conducted according to regulatory standards approved by the Peter MacCallum Cancer Centre Animal Ethics and Experimentation Committee. The following mouse strains were used for donor and recipient mice for transplantation C57BL / 6, PtprcaPepcb / BoyJ and NOD.Cg- / 7'% / c'''t% / 2 '", / b / / / SzJ (NSG). Lentiviral and retroviral production

[0244] For generation of SPLINTR lentiviral particles, 15 x 106HEK cells were plated into four T-175 tissue culture flasks. The next day, cells were transfected at 80% confluencewith SPLINTR-SR plasmid library, packaging plasmid (PSPAX2) and envelope (VSVG) using polyethylenimine ‘Max’ (PEI MAX) transfection reagent in Opti-MEM I Reduced Serum medium at a molar ratio 3:2:1. The supernatant containing viral particles was collected at 48 h and filtered through a 0.45 pm filter. Virus was concentrated 100 * at 3000g for 5 min using Amicon 15 mL Ultra- 15 filters (lOOkDa) (Millipore). SPLINTR virus was aliquoted at 10 pL and 20 pL, and stored at -80°C. Retroviral particles for overexpression of Xlr4b or empty vector were produced and concentrated as above with retroviral vector, packaging plasmid Gag -Pol, and VSVG used for transfection at a molar ratio 3:2:1.Preparation of SPLINTR barcoded cells for in vivo experimentation

[0245] For ex vivo expansion of mouse bone marrow cells, C57BL / 6 (CD45.2+) mice were humanely euthanized and bone marrow cells were collected from the femurs and tibias. Lineage positive cells were depleted using EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit (Stem Cell Technologies, #19856). Lineage negative HSPCs were then seeded in a 12-well fibronectin-coated plate in PVA-based media supplemented with TPO (100 ng / pl) and mouse SCF (10 ng / pl).

[0246] For barcoding, 1 * 106cells on 5-7 d of culture were seeded in a 24-well fibronectin-coated plate in 250 pl of PVA media supplemented with TPO (100 ng / pl) and mouse SCF (10 ng / pl). Each batch of virus was titrated to transduce cells at 2-10% reporter fluorescent protein expression, representing a multiplicity of infection of 0.02-0.1 to reduce the occurrence of multiple integrations per cell. Virus was added to cells and transduced by spinfection for 2 h at 2000g (no brake). After transduction, media was replaced and cultured as above. 48 h post transduction, cells were stained 1:500 with Propidium Iodide (PI, BD), 1 in 100 Lineage (Lin.) stain (APC, BD Bioscience, 558074, RRID:AB_1645213), 1 in 100 c-KIT (APC-Cy7, BioLegend, clone 2B8, 105826, RRID:AB_1626278) and 1 in 100 Sea I (PE, BioLegend, clone D7, 108107, RRID:AB_313344). Live cells were sorted for fluorescent protein expression and either Lin. negative or LSK using a FACS Aria Fusion flow sorter 3 and 5. The gating strategy for LSK is shown in Figure 1C. 3 x 104Lin. neg or 1 IO4LSK sorted cells (representing 2-3 * 103barcodes) were seeded in a 96-well plate for expansion in PVA-based media (supplemented as above).In vivo experiments using SPLINTR barcoded HSPCs

[0247] Barcoded cells were consistently expanded ex vivo for 4 weeks to obtain several million HSPCs, representative of 2-3 x 103clones. Each individual mouse received between 1-5 x 105HSPCs in transplantation. The remaining cells from the culture were cryopreserved for population-based genomic DNA barcode sequencing and subsequent baseline timepoint scRNA-seq. Mice were euthanized at defined timepoints and cells from the bone marrow, thymus and spleen were collected. Barcoded cell lineages were collected by flow cytometry as indicated below and lysed in Viagen for DNA barcoding as the end-point sample.In vivo experiment using Xlr4b-overexpressing HSPCs

[0248] For overexpression of Xlr4b in mouse bone marrow cells, 2* C57BL / 6 (CD45.2+) mice were humanely euthanized and bone marrow cells were collected from the femurs and tibias. Lineage positive cells were depleted using EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit (Stem Cell Technologies, # 19856). Lineage negative HSPCs were then seeded in a 12-well fibronectin-coated plate in PVA-based media supplemented with TPO (100 ng / pl) and mouse SCF (10 ng / pl).

[0249] For Xlr4b retrovirus transduction, 1 x 106BM cells on day 2 of culture were seeded in a 24-well retronectin-coated plate in 250 pL of PVA media supplemented with TPO (100 ng / pl) and mouse SCF (10 ng / pl). 30 pL of 100x concentrated empty vector or Xlr4b retrovirus was added to cells and transduced by spinfection for 1.5 h at 2000g (no brake). After transduction, media was replaced and cultured as above. On day 3, a second round of spinfection was performed with the same conditions, and cells were expanded for a further 48 h in PVA-based media supplemented as above.

[0250] On day 5 of culture, empty vector control or A / - / / >-ovcrcxprcssing cells were injected intravenously into PtprcaPepcb / BoyJ (CD45.1+) mice irradiated 8 Gy. Four replicate mice per condition each received 4xl05HSPCs. To assess thymic seeding and early engraftment of cells, BM, thymus and spleen tissue was collected at 3 weeks post transplantation. Dissociated tissues were analysed by flow cytometry for contribution of fluorochrome positive donor cells to tissue chimerism and lineage differentiation.Flow cytometry analyses

[0251] Cell analysis was performed on sorting was performed using a FACS Aria Fusion flow sorter 3 and 5 or FACS Aria II Sorter (BD Biosciences). Flow cytometry analysis was conducted on a Flow Fortessa (BD Biosciences). The following antibodies were used for flow cytometry on mouse cells and tissues: anti -mouse CD 117 (c-kit), APC-Cy7 (BioLegend, clone 2B8, 105826, RRID:AB_1626278); anti-mouse Ly6A / 5 (Sca-1), PE (BioLegend, clone D7, 108107, RRID:AB_313344); anti-mouse Lineage Antibody Cocktail, APC (BD Bioscience, 558074, RRID:AB_1645213); anti-mouse CD150 (SLAM), PE-Cy7 (BioLegend, TC15-12F12.2, 115914, RRID:AB_439797) anti-mouse CD3, PE-Cy7 (BioLegend, clone 17A2, 100220, RRID:AB_1732057); anti-mouse CD19, BV786 (BD, clone 1D3, 563333, RRID:AB_2738141); anti-mouse TER-119, APC (BioLegend, clone TER-119, 116211, RRIDA.B 313712); anti-mouse CD45.1, APC-Cy7 (Biolegend, clone A20, 110716, RRID:AB_313504); anti-mouse CD45.2, BV421 (BioLegend, clone 104, 109831, RRID:AB_10900256); anti-mouse CD8a, PE (BioLegend, clone 53-6.7, 100708, RRID:AB_312747); anti-mouse CD4, BV650 (BD, clone GK1.5, 563232, RRID:AB_2738083); anti-mouse TCRp, AF700 (BioLegend, clone H57-597, 109207, RRID:AB_313430); anti-mouse TCRy / 5, FITC (BioLegend, clone UC7, 13D5, 107503, RRID:AB_313312); anti-mouse Gr-1 (Ly-6G / Ly-6C), AF700 (BioLegend, clone RB6-8C5, 108422, RRID:AB_2137487); anti-mouse Gr-1 (Ly-6G / Ly-6C), PE (BioLegend, clone RB6-8C5, 108410, RRID:AB_313375); anti-mouse / human, AF700 (BioLegend, clone MI / 70, 101222, RRID:AB_493705); anti-mouse / human CDllb, PE (BioLegend, clone MI / 70, 101208, RRID:AB_312791). Flow cytometry data were analysed with FlowJo v.10.9 (Tree Star).Barcode reference library sequencing

[0252] The SPLINTR-SR library was sequenced by single-end 150 bp Illumina sequencing in technical duplicate to generate a reference library for downstream alignment and analysis. The sequencing reads were trimmed to remove constant flanking regions to extract the 60 bp SPLINTR barcodes sequences. Only sequences with an average Phred score of over 20 with the correct barcode structure were retained. Barcodes were then clustered into a consensus barcode library using Starcode (v.1.3). Any barcodes with less than five reads or not present in both technical replicate PCRs were excluded in the final reference library.SPLINTR barcode-seq and analysis

[0253] Barcode sequences were aligned to the reference library to generate a barcode counts table using BARtab vl.4. The unmapped reads were clustered and the barcodes with the correct sequence structure were added to the barcode counts table.

[0254] The barbieQ package in R was used to process and analyse barcode-seq data. Barcodes with a correlation coefficient greater than 0.95 were collapsed into clones by clustering to account for multiple integrations of barcodes into a single cell. The resulting collapsed clone counts table was filtered for clones in the top 90 to 99th percentile in at least one sample, ensuring the retained barcodes compose of at least 95% of all samples. Plots of the number of clones in each sample and the average contribution of each clone to samples were generated with ggplot2. The abundance of clones in each sample was visualised with plotBarcodeBubble from bartools or plotBarcodeHeatmap from barbieQ.

[0255] Correlation between samples were calculated by Pearson coefficients on log2 transformed counts per million (CPM) values for clones and plotted by ggplot2. The eulerr package in R was used to calculate overlaps in clones between conditions.

[0256] To calculate whether clones show biased lineage differentiation, the mouse samples were grouped into lymphoid or myeloid lineage and the testDiffProp function from barbieQ was used to calculate the LFC and significance of the lineage bias. Clones with an adjusted p-value of < 0.05 were classified as having significant lineage bias. Output of clones was calculated as the mean proportional contribution of that clone across all samples and are categorised as high (> 10%), medium (1 < 10 %) and low (< 1%). plotSamplePairCorrelation from barbieQ was used to plot pairwise Pearson correlation coefficients of clone CPM values between samples. Shannon diversity to measure the transcriptional heterogeneity of cells within a clone was calculated as described previously by Fennell et al. (2022, Nature, 601(7891): 125-131).scRNA-seq

[0257] scRNA-seq analysis of all samples was conducted using the 10X Genomics 3’ V3 Single Cell Gene Expression Library Preparation kit on a 10X Genomics chromium instrument. Cryopreserved HSPCs from ex vivo expanded culture or fresh BM were thawed rapidly at 37°C. Viable, propidium iodide negative and SPLINTR barcoded, VEX positive,cells were sorted using the BD Fusion 5 or 3. For time course experiment detailed in Figure 2D, cells were isolated fresh from culture without cryopreservation. Cells were labelled with TotalSeq Hashtag antibodies (BioLegend) as described by Fennell et al. (2022, supra). Samples were pooled and resuspended so that the final cell concentration was suitable for loading on to the 10X Chromium Single Cell Chip (1,500 cells / pL). Libraries were sequenced on the Illumina NovaSeq using paired-end 100-bp chemistry targeting 70,000 reads per cell. Hash tag libraries were sequenced on the NextSeq2000 using single-end 100-bp chemistry targeting -5,000 reads per cell.scRNA-seq analysis

[0258] Count matrices were generated from demultiplexed scRNA-seq fastq files using the lOx Genomics Cell Ranger (v.7.0.0) count pipeline against the mmlO genome. scRNA-seq quality control was performed using Seurat v.4 in R. Low-quality cells were removed by filtering out cells that had fewer than 500 genes or 1,000 unique molecular identifiers (UMIs). Cells with greater than 10% mitochondrial RNA content were also removed. Multiplexed scRNA-seq samples containing TotalSeq-A hashtag oligo information were demultiplexed using the CITE-seq-count v.1.4.5 and HTODemux methods within Seurat v.4 using the default settings. Normalisation of scRNA-seq datasets was performed using the SCTransform method with regressing out the percentage mitochondrial reads.

[0259] Cell cycle phase assignments were generated per cell from scores derived using the CellCycle Scoring function within Seurat, using cell cycle genes as previously described in Fennell et al. Dimensional reduction, k-nearest neighbour graph construction and clustering were performed using scanpy in python. The first 50 principal components were used to compute a nonlinear dimensional reduction using the UMAP method. Leiden clustering was performed at different resolutions to cluster cells. Differential gene expression analysis for clusters and groups of cells was performed using MAST in Seurat. Volcano plots of differentially expressed genes were generated by enhanced volcano in R. The engraftment score was calculated with the AddModule Score function in Seurat using the top 10 most significantly up-regulated genes between the engrafting and non-engrafting cells in the HSPC cluster.

[0260] Velocyto was used to calculate RNA velocity, providing insights into the dynamic state of gene expression. Subsequently, CellRank was used to generate velocity stream plots and perform random walk analyses.

[0261] Differential gene expression analysis was performed using MAST to identify the most significantly upregulated genes in Group 1 clones compared to Groups 2, 3, and 4 clones. For each pairwise comparison (Group 1 vs. Group 2, Group 1 vs. Group 3, and Group 1 vs. Group 4), the top 100 most significantly upregulated genes were selected based on adjusted p-value. Genes that were consistently upregulated were identified from at least two of the three comparisons, resulting in a set of 54 genes. To further characterise the importance of these genes, logistic regression was performed using the tidymodels framework in R. A multivariate logistic regression model was fitted using the 54 selected genes as predictors. The model coefficients were calculated for each gene, providing insights into their relative importance in distinguishing Group 1 clones from the other groups.Example 2Engraftment, clonal output and lineage commitment are cell-intrinsic properties of transplanted HSPCs

[0262] The molecular sequelae of ex vivo murine HSPC expansion was characterized by serial sampling the culture over 5 weeks (Figure 1A). A -1000 fold expansion was observed regardless of whether the starting material were LSK cells (Lineage- Scal+ c-Kit+) or Lineage negative (Lin-) HSPCs derived from the bone marrow (Figure IB). Immunophenotypically, the culture remained largely undifferentiated with >80% of cells remaining Lin- (Figure 1C). To gain greater granularity into cellular states, single cell RNA sequencing (scRNA-seq) of serial samples from the culture was performed. The culture originates with cells expressing transcriptional signatures of stem and early progenitors and with time there is progressive increase in cells expressing lineage specific programs (Figures ID and IE), despite no major change in immunophenotype.

[0263] Having established the baseline consequences of ex vivo HSPC culture, HSPC behaviour post-HSCT was assessed at single clone resolution by employing a new lineage tracing system called SPLINTR-X (Figure 2A), which enables transcriptomic clonal information to be ascribed in HSPCs. Resolving cell-intrinsic properties from external influences requires the concurrent monitoring of clonal behaviour across multipleindividuals. To address this issue, barcoding was coupled with a clone splitting strategy. In this method, each HSPC was labelled with a unique barcode at the start of the expansion culture to provide a heritable clonal identity (Figure 2D). Subsequent serial expansion ex vivo means that each HSPC clone will give rise to multiple progeny. One-third of the culture was harvested after 3 weeks, evenly split and transplanted into several recipients after sublethal (4Gy) irradiation. Every recipient received 1 x 105cells with identical clonal representation. The remaining culture was then perpetuated until 5 weeks when again it was evenly split with one-third of the culture being transplanted (5 x 105cells) into several recipients and the remaining culture used to assess baseline clonal structure prior to transplantation (Figure 2D). Thus, every mouse received an identical population of HSPC clones from the culture, which allowed for an assessment of whether clonal behaviour is a cell-intrinsic heritable property and how extended culture altered clonal fate.

[0264] Using sub-lethal irradiation and transplantation of 1 x 105cells (derived from 2,768 clones), haematopoietic reconstitution was clearly established by 4 weeks. Surprisingly however, <1% of the transplanted clones were able to engraft and contribute to haematopoiesis (Figure 2E). Haematopoietic reconstitution was comparable between Lin-and LSK cultures and was not compromised by a longer duration of culture (Figure 2F). When behaviour was assessed across all recipients there were two striking findings: first, although every clone that engrafted had unique clonal behaviour, this behaviour was identical in every transplant recipient. For example, if a clone was capable of multi-lineage reconstitution, it behaved as such in every recipient. However, if a clone was restricted to lymphoid or myeloid reconstitution this behaviour was also identical in every recipient (Figure 1G). Second, clonal output, which is a measure of the amount each clone contributes to a particular cell lineage, was also highly concordant in all transplant recipients (Figure 1G and Figure 3C). Remarkably, the only exception to this concordance in both clone fate and output was the commitment and production of T cells (Figures 2G and 2H).

[0265] To understand how the environmental variables introduced by extended culture altered cell behaviour clones that engrafted after either 3 or 5 weeks of culture were considered. Interestingly, the majority of clones that engrafted after 5 weeks of culture were also the same clones that also engrafted after 3 weeks (Figure 21 and Figure 3D). Notably, these shared clones had the highest clonal output contributing to the vast majority of the haematopoietic cells derived from the transplanted cells at both time points. Moreover, the proportional clonal output measured in the bone marrow of recipient mice remained highlycorrelated regardless of the duration in culture (Figure 2J). Together, these data emphasise that each HSPC has a highly cell-intrinsic and heritable cell fate and output, and this behaviour is not dramatically influenced by environmental factors ex vivo. Moreover, unlike the reconstitution of myeloid, erythroid and B-cell lineages, rapid T cell reconstitution demonstrates more variable clonal kinetics, suggesting that T cell reconstitution has unique challenges that extend beyond HSPC engraftment.Example 3Changes in clonal output cater for physiological need in HSCT recipients

[0266] To assess how an allogeneic and altered immune microenvironment with marked variations in stroma, endothelium and cytokines influences clonal behaviour, LSK cells were harvested from the bone marrow of C57BL / 6 mice, before being barcoded and expanded for 5 weeks. 2.5 x 105cells (derived from 2064 clones) were then transplanted into syngeneic / autologous (C57BL / 6) and allogeneic (NSG) recipients (Figure 4A). Consistent with the data presented in Example 1, <1% of the transplanted clones engrafted in either the autologous or allogeneic recipients (Figure 5A). Notably, 80% of the clones that engrafted in the autologous mice also engrafted in the NSG setting, and it was these shared clones that had the highest output collectively contributing to >90% of the haematopoietic reconstitution derived from the transplanted HSPCs (Figure 4B).

[0267] A detailed clonal assessment revealed that, aside from T-cells, clonal cell fate commitment and lineage bias was similar in all engrafting clones across both C57BL / 6 and NSG recipients (Figure 4C and Figure 5B). Whilst these data reinforced the remarkable cell-intrinsic feature of HSPC engraftment and lineage bias, clonal output was more varied and showed three major patterns. About half of all engrafting clones not only shared lineage bias but also had a similar clonal output in both C57BL / 6 and NSG recipients. The remaining clones could be split into a group of myelo-erythroid biased clones that had a higher output in autologous C57BL / 6 recipients following sub-lethal irradiation and a group of lymphoid-biased clones that had a higher output in the NSG recipients that lacked an adaptive immune system (Figures 4C and 4D). To identify properties of the clones that demonstrated the capacity to sense and respond to a physiological need, the pre-transplant transcriptional program of the clones with identical behaviour in allogeneic and autologous recipients were compared with those that preferentially increased their output in NSG or irradiated C57BL / 6 mice. The clones with higher output in NSG were those that prior to transplantation showedfeatures of lymphoid-bias and expressed higher levels of genes such as Gml5987 and Gprl83 (Figures 5C and 5D). In contrast, the pre-transplant program that distinguished clones with enhanced myelo-erythroid output in the irradiated C57BL / 6 recipients showed increased expression of Cleclla / Scgf and Nuprl (Figures 5E and 5F), which have experimentally been validated to be regulators of enhanced HSPC engraftment in inflammatory microenvironments, including following radiation conditioning (see, e.g., Ramalingam et al., 2020, Nature Communications, 11(1): 666; and Wang et al., 2022, Haematologica, 107(1): 154-166).

[0268] These striking findings provides further context to recent work describing a physiological need-sensing process that directs HSPC cell fate after HSCT (Calabria et al., 2024, Nature, 636(8041): 162-171). The initial interpretation of this study was that the bone marrow microenvironment of recipients with a particular cytopenia re-purposes the cell fate potential of up to half of engrafting HSPC to preferentially replenish the deficient lineage. Interestingly, the data presented herein also shows that up to half of engrafting clones were able to sense and respond to physiological need. However, rather than altering the cell fate potential of the engrafting HSPC, physiological need was catered for by enhancing the output of clones with the cell-intrinsic lineage bias that most suits the need of the host.Example 4A transcriptional signature that facilitates engraftment and high output

[0269] The transcriptional landscape of barcoded HSPCs was examined by scRNA-seq prior to transplantation in four independent experiments. Briefly, barcoded clones were independently expanded for 4 weeks prior to transplantation into multiple C57BL / 6 recipients irradiated 4Gy. At 4 weeks post HSCT, haematopoietic lineages were isolated from bone marrow, spleen and thymus to identify the clones that contributed to erythroid, myeloid and lymphoid engraftment (i.e., contributed to >1% of any haematopoietic lineage). Consistent with Figures 2E and 5A, only a very rare subset of transplanted clones made a meaningful contribution to rapid haematopoietic engraftment (Figure 6A).

[0270] Prior efforts to identify the transcriptional programs that dictate engraftment have not had the benefit of a clone splitting approach, which allows for the examination of the precise transcriptional program of a clone prior to transplantation, and correlating this with a specific and reproducible clonal phenotype across multiple recipients. Therefore,scRNA-seq was used to compare the baseline transcriptome of the engrafting clones with clones that also express the established HSPC signature but fail to engraft. A major discriminating feature of engrafting clones was that their progeny exhibited strong transcriptional memory and retention of the gene expression program following cell division (Figure 4E). In contrast, the progeny of non-engrafting clones showed marked transcriptional drift. This heritable behaviour cannot be easily explained by proliferative capacity ex vivo, as the number of cells produced per clone (clone size) was comparable (Figure 6C). Single cell trajectory analyses using either RNA velocity or diffusion pseudotime, which leverages random walk approaches, both highlighted the recursive behaviour exhibited by engrafting HSPCs, further emphasising that the cell-intrinsic properties of engrafting clones are distinctive (Figure 6C).

[0271] To identify the distinguishing transcripts between engrafting and non-engrafting HSPCs, differential gene expression analyses was confined to cells within the HSPC cluster in one replicate experiment (Figure 4F). This identified several genes that were either more or less prominently expressed in engrafting clones. Whilst some genes such as Mecom and Ly6a are well established markers of stem cell potential, most of the identified genes were uncharacterised in the context of haematopoiesis.Example 5Increase intensity of conditioning increases clonal output, but not engraftment

[0272] Underpinning the success of HSCT are conditioning therapies, which serve several purposes: (1) to enhance disease eradication; (2) to cause transient immunosuppression reducing graft rejection; and (3) to create space for the engraftment of donor HSPCs (see, e.g., Zulu et al., 2017, “Principles of Conditioning Therapy and Cell Infusion” in The European Blood and Marrow Transplantation Textbook for Nurses, Springer). Reduced intensity conditioning is frequently used in less fit individuals to reduce the risk of transplant related morbidity, but the consequence of this choice on haematopoietic reconstitution and HSPC behaviour remains unexplored. To address this issue, an identical pool of HSPC clones was transplanted into mice that had received either 4Gy or 8Gy of conditioning total body radiation (TBI) and followed haematopoietic reconstitution over 16 weeks (Figure 7A). Unexpectedly, these data revealed that more intensive conditioning did not increase the number of clones able to engraft the recipients (Figure 7B), instead clonal output from the engrafting clones was markedly increased for erythroid, myeloid and B-celland T-cell reconstitution (Figure 7C). Notably this increase in clonal output is not shortlived and is maintained for at least 16 weeks post-conditioning.Example 6The thymus is the most instructive microenvironment for HSPCs

[0273] An identical population of barcoded HSPCs were transplanted intravenously (IV) into the tail vein or directly, via ultrasound guidance, into the thymus. As shown in Figure 8A, all cell-intrinsic lineage potential and bias of the HSPCs were restricted by the thymic microenvironment resulting in the sole production of T-cells. Notably, T-cell chimerism of donor cells increased by ~5-fold following intra-thymic transplantation (ITT) (Figure 8B) and intra-thymic T-cell development proceeded normally leading to typical egress into the peripheral circulation (Figures 12C and 12D).

[0274] An identical population of HSPC clones derived from C57BL / 6 mice were expanded and transplanted via ITT into unconditioned NSG mice or C57BL / 6 mice who had received a variety of conditioning therapies (Figure 7D). As an additional control, an identical population was also transplanted into C57BL / 6 mice via IV injection after 4Gy conditioning.

[0275] ITT of HSPCs into NSG led to a rapid regrowth of the thymus and, despite the fact that the thymic stroma has never produced T-cells previously, it was similarly instructive for cell fate by not allowing any escape of HSPCs to other haematopoietic niches within the body and generating only T-cells (Figures 8E and 8F). The thymic regrowth following ITT of HSPCs in NSG mice was not comparable to the thymic size of an aged matched C57BL / 6 thymus after ITT (Figures 8G). However, a wholly unexpected finding is that the atrophied thymic stroma of the unconditioned NSG mice was able to support engraftment of more than double the number of clones which engrafted the unconditioned C57BL / 6 mice and a comparable number of clones which engrafted C57BL / 6 mice following conditioning with chemotherapy or varying doses of radiotherapy (Figures 7E and 7F). These data further illustrate that whilst conditioning therapy is required for efficient engraftment, neither the intensity nor modality of conditioning influences the number of clones able to engraft. Finally, identical conditioning therapy resulted in 3-fold greater engraftment of clones capable of T-cell production with ITT compared to IV transplant, underlining the fact thatthymic seeding is the major rate limiting step for T-cell reconstitution regardless of conditioning therapy (Figures 7E and 7F).Example 7HSPCs show no inherent bias to specific T cell subsets but have distinct kinetics of differentiation

[0276] In the thymus, HSPCs are faced with a myriad of fate choices that are governed by receptor-ligand interactions and signaling events within a cytokine rich milieu that directs differentiation. One of the earliest choices available to HSPCs is the bifurcation point to commit to either y8 or a(3 lineage fate. There are two-models that have been put forward to explain this choice (i) a pre-commitment-selection model and (ii) a TCR-signal strength model, which has more support (see, e.g., Fahl et al., 2018, “TCR Signaling Circuits in oc(3 / yS T Linage Choice” in Signaling Mechanisms Regulating T Cell Diversity and Function, CRC Press / Taylor & Francis). As this open question has not been studied at clonal resolution for individual HSPCs within the thymus, HSPCs were barcoded and expanded, followed by ITT and harvested the thymus and spleen for analysis of T-cell lineage commitment 4 weeks after transplant (Figure 9A). T-cell lineages harvested from spleen, lymph nodes and blood were highly correlated, suggesting that clones have equal tissue tropism (Figure 9B). These data showed that the vast majority of HSPCs are not skewed towards a y6 or a(3 lineage fate and instead they produce both equally (Figure 9C). Moreover, once committed to the a(3 fate they were shown to be equally capable of undergoing positive selection by engaging with either MHC-I or MHC-II. Notably, a minor population of HSPCs (<8%) that has a strong contribution to y6 T-cells was identified, suggesting that whilst pre-commitment may occur, this occurs only in a minor population of HSPCs. Moreover, while every HSPC clone was capable of producing y6 or a(3 T-cells, clonal analysis revealed that not all clones progressed through lineage commitment with the same kinetics. Overall, some HSPCs undergo rapid positive and negative selection within the thymus and emigrate to peripheral tissues, whereas as other HSPCs have much slower kinetics of differentiation (Figure 9D). Notably, clones with higher output in peripheral y6 T-cells compared to a(3 T-cells demonstrated slower differentiation kinetics in the thymus, indicative of the faster kinetic recovery of y6 lineage post donor cell engraftment.

[0277] HSPC are most frequently transplanted via IV injection and subsequently home to the bone marrow prior to seeding the thymus. Therefore, it is possible that this processmay be necessary for the functionally integrity of T-cells and ITT, which bypasses this process, may not produce functionally equivalent cells. To formally test this, an identical population of expanded and barcoded HSPCs was transplanted either IV or via ITT in multiple mice (Figure 9E). Four weeks following transplant, naive T-cells were isolated from the spleen and activated in vitro. Naive T-cells produced after either ITT or IV transplant were equally adept at differentiating to central memory or effector cells (Figure 9F). When assessed at clonal resolution, ITT of HSPCs resulted in more clones contributing to T-cell production, but each clone was equally capable of being activated and differentiating proportionately (Figures 9G and 9H). To extend these findings, HSPCs were obtained from transgenic mice expressing a P 14 T-cell receptor (TCR) specific for an immunogenic peptide (H-2Db-gp33) derived from the lymphocytic choriomeningitis virus (LCMV). These HSPCs were used for ITT and after 4 weeks naive T-cells were harvested from the spleen and adoptively transferred into animals infected with LCMV (Figure 91). T-cells produced from ITT were functionally intact and capable of producing a robust response to infection. Taken together, these data demonstrate that every HSPC that seeds the thymus produces y8, and «P T-cells that are CD4+ or CD8+. Moreover, homing to the bone marrow prior to thymic seeding is not required for the functional competency of T cells.Example 8Thymic seeding is the rate limiting step to T cell reconstitution following HSCT

[0278] Following HSCT, a transplanted HSPC has several bottlenecks to negotiate prior to contributing to haematopoiesis. An HSPC first need to home to the bone marrow, which is the primary site of production for all haematopoietic lineages except T cells. In the case of T cells, there is an additional requirement to seed the thymus to prior to lineage commitment. To understand how each of these potential bottlenecks influence HSPC potential, an identical population of expanded and barcoded HSPCs were transplanted into three different sites: (i) IV, as this is the most frequent route of administration; (ii) intrafemorally (IF) to bypass the requirement for homing to the bone marrow and; (iii) via ITT, to bypass both homing and thymic seeding (Figure 10A). The number of clones able to engraft a recipient following either IV or IF transplant were the same (Figure 11 A). Moreover, clonal potential with regards to cell fate and clonal output was indistinguishable regardless of whether the engrafting HSPCs were delivered via the IF or IV route (Figure 1 IB). These data, at clonal resolution, indicate that homing to the bone marrow is not a major rate limiting step to engraftment or lineage commitment. Moreover, by serially followingthe behaviour of these clones over 8 weeks, the majority of unbiased and lymphoid biased clones maintained this behaviour throughout whereas most myelo-erythroid biased clones at 4 weeks evolved to have a more balanced contribution to haematopoiesis by 8 weeks (Figure 11C).

[0279] Compared to IV or IF transplantation, when the identical clonal population was delivered via ITT, up to 3-times as many HSPC clones were able to engraft the thymus, all contributing to T cell production (Figure 10B and Figure 9D). Interestingly, when clones that contribute to T cell production were directly compared across the different transplant routes, there was a high correlation between clonal fate and output for HSPCs delivered either IV or IF. Conversely, this correlation was markedly attenuated when these two routes of transplant are compared to ITT of HSPCs (Figure 10C and Figure 9E). Although the HSPC clones that give rise to T cells after IV or IF transplant also give rise to T cells after ITT, there are many HSPC clones that only engraft and give rise to T cells when directly transplanted into the thymus (Figure 10C and Figure 9E). Interestingly, when these clones that only engraft via ITT are delivered either IV or IF, they fail to engraft and contribute to any haematopoietic lineage (Figure 10C, Group 4 clones). These data demonstrate that not only is the thymus the most instructive microenvironment, but it is also the most accommodating microenvironment rescuing HSPCs that would otherwise have failed to engraft.

[0280] Following either IV or IF transplantation, approximately half of the HSPC clones that give rise to T cells do so in in replicate mice. In contrast, when delivered via ITT approximately 90% of clones are present in replicate mice (Figure 10D). Here, for each clone detected in replicate mice, the proportion of the clonal population present within the thymus was quantified at the earliest stage of T cell commitment (DN) through to the intermediate (CD3-DP and CD3+DP), mature stages (a[3 SP and y<5 T-cells) and mature (a[3 SP) T cells egressed to the spleen. When HSPCs were transplanted directly into the thymus, some clones progressed through T cell commitment quickly, whereas others had a slow rate of differentiation. However, in replicate mice the kinetics of differentiation for each individual clone was observed to be strikingly similar. In contrast, when HSPCs were delivered via IV or IF transplantation, there was a lower correlation in the kinetics of differentiation for individual clones between replicate mice, likely reflecting the periodic and gated waves of thymic seeding (Figure 10D). Taken together, these data demonstrate that in addition to having cell-intrinsic mechanisms that determine cell fate and clonal output, every HSPCwhich produces T cells also has a cell-intrinsic clock which determines the kinetics of T cell differentiation. Although different for each clone, this clock is synchronised and initiated when a HSPC seeds the thymus, thereby emphasizing that homing to the bone marrow is not a constraint to lineage commitment, Rather, the major bottleneck to rapid T-cell reconstitution is successful thymic seeding.Example 9A HSPC transcription program associated with robust T cell reconstitution

[0281] A granular analysis of HSPC clonal behaviour following transplantation into the three different microenvironments revealed several clones with four distinct outcomes (Figures 10E and 10F):

[0282] Group 1 consisted of clones with multilineage potential but with a lymphoid bias. Importantly, these clones were equally adept at producing T cells with high clonal output regardless of the route of transplantation.

[0283] Group 2 contained multilineage clones that when transplanted IV or IF were myelo-erythroid biased and capable of producing B cells but barely contributed to T cell production. Following ITT, these same clones were entirely capable of T cell production albeit with low output.

[0284] Group 3 comprised high-output clones with a strong lymphoid bias that largely only produced B-cells when transplanted IV or IF but produced a significant proportion of T-cells following ITT.

[0285] Group 4 were made up of low-output HSPC clones that failed to engraft after IV or IF transplant but engrafted the thymus and produced T-cells following ITT. Whilst the clones that only engraft after ITT were capable of producing T cells, their overall contribution to T-cell production is markedly lower compared to the clonal output of Group 1 clones.

[0286] Overall, Group 1 represented the HSPC clones with the ideal cell-intrinsic program for engraftment, thymic seeding and high output resulting in robust T cell reconstitution after HSCT. By contrast, the Group 2, 3 and 4 clones were all capable of producing T cells when transplanted into the thymus, but had cell-intrinsic programs thatmanifested in a failure to engraft and / or rapidly seed the thymus to strongly produce T cells when they were transplanted either IV or IF (Figures 10E and 10F).

[0287] When the transcriptomes of each Group were projected onto a UMAP and compared with transcriptome of cells within the differentiation hierarchy for haematopoiesis, all four groups were shown to have a substantial proportion of cells which display the transcriptional signature ofHSPCs (Figure 12A). Hierarchical clustering showed that while clones within Groups 1-4 were intermingled, they were largely separated from the non-engrafting clones by the stronger maintenance of HSPC transcriptional programs, and a low output of cells expressing myeloid progenitor transcription programs (Figure 12B). A number of non-engrafting clones also had cells with lymphoid transcription programs, indicating that lymphoid differentiation in vitro does not predict lymphoid potential and fate in vivo.

[0288] To pinpoint the unique transcripts distinguishing Group 1 clones from Groups 2-4, differential gene expression analysis was focused on cells from each Group within the HSPC cluster (Figure 12C). This approach revealed a consistent gene expression signature that distinguished the high-output T cell producing clones in Group 1 from clones that produced only myeloid and B-cells (Group 2), exclusively produced B-cells (Group 3) or failed to engraft (Group 4) after IV transplantation. This novel composite gene set, composed largely of genes with unknown function in both HSPCs and T cell development.

[0289] Unlike well-established genes associated with lymphopoiesis such as Flt3, Satbl, Il7r, Dnii. which show a discernible increase in expression coincident with lymphoid commitment, many of the genes specifically upregulated in Group 1 cells showed a pattern of expression that is more widespread including in stem cells and lymphoid progenitors. Although the majority of the genes expressed more prominently in Group 1 clones remain largely uncharacterized in the context of haematopoiesis and more specifically T-lymphopoiesis, a noteworthy gene that discriminated Group 1 clones from the other groups in every differential expression analysis 'wasXlr4b, which is of unknown molecular function (Figure 12C and Figure 13 A).

[0290] Since Xlr4b demonstrated expression in a rare subset of clones in adult HSPCs, expression of Xlr4b may represent a remnant developmental pathway to produce T-cells. This is reflected in the data presented in Figure 15, where -12% of bone marrow HSPCs and-30% T-cells were shown to express Xlr4b at 16.5 days of development with a progressive decline in expression by birth (P0). Thus, Xlr4b may be highly relevant to the fetal T cell program in establishing the adaptive immune system.

[0291] To understand if expression of Xlr4b has a role in lymphoid commitment and output, Xlr4b was ectopically expressed in expanded C57BL / 6 (CD45.2) HSPCs and transplanted cells into 8Gy irradiated CD45.1 / Ly5.1 / Ptprcarecipients (Figure 14A). The bone marrow chimerism of Xlr4b expressing cells was reduced despite high levels of engraftment of non-transgenic donor cells, suggesting that the association of Xlr4b with enhanced T-cell production cannot be attributed to increased HSPC engraftment (Figures 14B and 14C). Increased Xlr4b expression was shown to markedly reduce B-cell differentiation (Figure 12D). Xlr4b expressing HSPCs were also very adept at seeding the thymus, and underwent normal T cell commitment and selection (Figures 12E-12F). This functional evidence highlights a role for this completely uncharacterised gene in preferentially regulating T-cell commitment and production following haematopoietic stem cell transplantation.

[0292] The rapid change in lymphoid differentiation suggested that Xlr4b has a direct role in modulating lymphoid fate decisions in the bone marrow. To understand the mechanism associated with ectopic expression of Xlr4b. the kinetics of bone marrow engraftment of HSPCs delivered intravenously were studied at 24 hrs, 72 hrs, Wkl, Wk2, Wk 12 post engraftment. Xlr4b did not influence the homing or early expansion of transplanted HSPCs in the bone marrow with similar numbers of transplanted HSPCs and proliferation kinetics (Figures 16A and 16B). In addition, Xlr4b expression did not bypass bone marrow homing, leading to the direct seeding of the thymus from intravenous injection (Figures 16C and 16D). By Wkl post transplantation, Xlr4b expression impaired B-cell production, which was maintained during longer-term bone reconstitution (12 weeks), suggesting both a rapid and durable function in lymphoid development.

[0293] To explore the mechanism by which Xlr4b regulates lymphoid output in the bone marrow, single cell RNA-sequencing was performed on lineage negative HSPCs at Wkl, Wk2, Wkl2 post-transplantation. At Wkl, annotation of stem and progenitor cell populations showed reduced numbers of CLPs in the bone marrow (Figure 17A) with a concomitant (Figure 17B) reduction in B cell differentiation and maintained T cell seeding of the thymus (Figure 17C), supporting a role for Xlr 4b in rapidly influencing B versus T-cell fate decisions. Subsequently, as multilineage reconstitution became established over time the number of CLPs normalised, demonstrating that differentiation of multipotent stem and progenitor cells to CLPs was unaffected. However, differentiation of CLPs to committed B-cell progenitors (PrB) was reduced (Figure 17D). Single cell regulatory network inference in CLPs identified that Xlr4b expression significantly reduced gene regulatory networks controlled by key B-cell transcription factors, supporting a role for Xlr4b expression in regulating bone marrow lymphoid fate at the level of CLP differentiation (Figure 17E). Therefore, expression of Xlr4b likely regulates the key transition point in bone marrow lymphoid differentiation, where common lymphoid progenitors commit to B or egress the bone marrow to seed the thymus.

[0294] The FAM9 family of genes are the human orthologs of Xlr genes, with FAM9B likely representing the human ortholog of Xlr 4b. To understand if FAM9B might also function in lymphoid development, FAM9B cDNA (SEQ ID NO: 24) was overexpressed in transplanted mouse HSPCs. Similar to ectopic expression of Xlr4b. FAM9B expression markedly reduced B-cell differentiation while enabling thymic seeding and reconstitution (Figure 18).

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An engineered haematopoietic stem or progenitor cell (HSPC), which has been modified to ectopically express an X-linked lymphocyte -regulated (Xlr) gene or an ortholog thereof relative to an equivalent unmodified HSPC.

2. The engineered HSPC of claim 1, wherein the Xlr gene is selected from the group consisting of Xlr 4a and Xr 4b.

3. The engineered HSPC of claim 1, wherein the ortholog is selected from the group consisting of FAM9A, FAM9B and FAM9C.

4. The engineered HSPC of claim 3, wherein the ortholog is FAM9B.

5. The engineered HSPC of any one of claims 1-4, comprising an exogenous nucleic acid construct encoding the Xlr gene or an ortholog thereof.

6. The engineered HSPC of claim 5, wherein the exogenous nucleic acid construct has been introduced by gene editing, homologous recombination, non-homologous end joining, transfection or transduction.

7. The engineered HSCP of claim 5 or claim 6, wherein the exogenous nucleic acid construct comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 70% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

8. A composition comprising a population of the engineered HSPC of any one of claims 1-7.

9. The composition of claim 8, further comprising a pharmaceutically acceptable carrier.

10. A nucleic acid construct comprising a nucleotide sequence encoding an Xlr gene or an ortholog thereof.

11. The nucleic acid construct of claim 11, comprising a nucleotide sequence selected from any one of SEQ ID NOs: 1-20, or a sequence having at least 70% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 1-20.

12. A vector comprising the nucleic acid construct of claim 10 or claim 11.

13. A method for modifying an HSPC to ectopically express an Xlr gene or an ortholog thereof, wherein the method comprises:(a) contacting an isolated HSPC or a population thereof, with the nucleic acid construct of claim 10 or claim 11, or the vector of claim 12; and (b) culturing the HSPC of step (a) in vitro or ex vivo for a time and under conditions suitable to ectopically express an Xlr gene or an ortholog thereof relative to an equivalent unmodified HSC.

14. The method of claim 13, further comprising inducing T cell or T cell-like differentiation following step (b).

15. A method for enhancing T cell reconstitution following haematopoietic stem cell transplantation, the method comprising administering an effective amount of the engineered HSPC of any one of claims 1-7, or the composition of claims 8 or claim 9 to a subject in need thereof.

16. The method of claim 15, wherein the endogenous haematopoietic compartment of the subject has been depleted prior to the administration of the engineered HSPC or the composition.

17. The method of claim 16, wherein the endogenous haematopoietic compartment has been depleted by chemotherapy or radiotherapy.

18. The method of any one of claims 15-17, wherein the subject has a haematological disease.

19. The method of any one of claims 15-18, wherein:(a) the engineered HSPC, or the composition comprises HSPC that are autologous to the subject; or(b) the engineered HSPC, or the composition comprises HSPC that are allogenic to the subject.

20. A method for the treatment of a haematological disease, the method comprising administering the engineered HSPC of any one of claims 1-7, or the composition of claims 8 or claim 9 to a subject in need thereof.

21. Use of the engineered HSPC of any one of claims 1-7, or the composition of claims 8 or claim 9 in the manufacture of a medicament for the treatment of a haematological disease.

22. The method of any one of claims 18-20, or the use of claim 21, wherein the haematological disease is selected from the group consisting of multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelofibrosis, essential thrombocytosis, polycythemia vera, solid tumors, aplastic anemia, severe combined immune deficiency syndrome (SCID), thalassemia, sickle cell disease, chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anaemia, Blackfan-Diamond anaemia, enzymatic disorders, non-malignant autoimmune diseases, neuromyelitis optica and multiple sclerosis.

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