cell

Abstract

The present invention relates generally to the fields of oncology and molecular biology. More particularly, it concerns allogenic CD7 CAR T-cells for targeting and treatment of CD7+ diseases, such as CD7 positive malignancies. Included within the scope of the invention are engineered donor cells, and nucleic acid constructs as well as miRNAs and VHH sequences useful for performing the invention.

Description

[0001] CELL

[0002] BACKGROUND OF THE INVENTION

[0003] Field of the Invention

[0004] The present invention relates generally to the fields of oncology and molecular biology. More particularly, it concerns allogenic CD7 CAR T-cells for targeting and treatment of a CD7+ disease, such as CD7 positive malignancies.

[0005] Description of Related Art

[0006] CD7 (cluster of differentiation 7) is a transmembrane glycoprotein and cell surface costimulatory molecule. CD7 appears early in T-cell ontogeny and plays an important role in T-cell and T-cell / B-cell interactions during early lymphoid development, being expressed on human T-cells and natural killer (NK) cells, as well as on cells in the early stages of T-cell, B- cell, and myeloid cell differentiation. In CD8+T-cells, CD7 can be used to define distinct subsets of cells, or as a marker of CD8 T-cell differentiation. This corresponds to three populations: CD7 high, low, and negative cells, which in turn translate into subsets of cells with distinct functional and phenotypic properties. It has been reported that the P-galactoside- binding lectin galectin-1 binds CD7, inducing apoptosis of thymocytes and T-cells, with implications for T-cell malignancies amongst other diseases.

[0007] CD7 is expressed in more than 95% of acute T-cell leukaemia (T-ALL) and NK / T-cell lymphomas (TCLs), acute myeloid lymphoma and certain types of peripheral T-cell lymphomas. This makes CD7 an attractive therapeutic target in T-cell malignancies, with normal CD7 expression mainly confined to T-cells andNK cells, reducing the risk of off targetorgan toxicity.

[0008] T-cell malignancies are a heterogeneous group of disorders of clonal growth and T-cell dysfunction broadly grouped into TCLs and T-ALLs. Initial treatment with conventional chemotherapy often results in profound toxicity with insufficient efficacy to sustain remission. Despite some progress, the need remains for new, targeted regimens to improve patient outcomes. Chimeric antigen receptor (CAR) T-cells are among the most promising immunotherapies for cancer. Due to the similarities between B- and T-lymphoid malignancies, extending CAR T- cell therapies to T-cell malignancies appears promising.

[0009] Adoptive cell therapy (ACT), and in particular the use of T-cell receptor (TCR) engineered and CAR T-cells, holds great promise for the treatment of cancer, and certain infectious and autoimmune diseases. Extremely high cure rates have been reported for certain cancers, specifically haematological malignancies (Jackson et al., 2016), while limited success has been seen with other malignancies, specifically solid tumours (O’Hara, 2016; Han et al., 2017; Irving et al., 2017). Many of these failures are attributed to a hostile tumour microenvironment that provides physical, molecular and immunosuppressive barriers for engineered immune cells to overcome. Likewise, to date, constructs that can be used for stimulating an immune cell activity, or inhibiting immune checkpoints have not been developed.

[0010] Applying CD7-based CAR T-cell therapies in practice is problematic as the target antigen is shared between healthy and malignant T-cells. This shared antigenicity can cause fratricide in CAR-transduced T-cells as internalization of CD7 from the T-cell surface following CAR expression is incomplete, inhibiting their manufacture and viability. It may also then result in eradication of normal peripheral T-cells after administration, leading to severe immune deficiency. Such an on-target / off-tumour effect would be more profound and less treatable than is found after depletion of normal B-cells with e.g., CD19 CAR T-cell-based therapies.

[0011] It has been shown that targeted genomic disruption of the CD7 gene prevents this fratricide and enables expansion of CD7 CAR T-cells without compromising their cytotoxic function. CD7 CAR T-cells have similarly been shown to provide robust cytotoxicity against malignant T- cell lines and offer protection in a mouse xenograft model of T-ALL.

[0012] Allogeneic CAR T-cells, generated from healthy donor T-cells, can provide ready-to-use, blast- free therapeutic products but may require further modifications to prevent graft-versus-host disease and rejection by the recipient’s immune cells. In the past allogeneic CAR T-cells have been created using gene editing techniques that induce DNA double- stranded breaks, resulting in unintended on-target editing outcomes with potentially unforeseen consequences.

[0013] CAR T-cell therapy is an established form of treatment for B-cell malignancies and myeloma, and regulatory approval against other cancers is expected. The field is progressing rapidly as improvements are made in treatment safety, efficacy and feasibility of these therapies. SUMMARY OF THE INVENTION

[0014] In a first aspect, the present invention provides an engineered donor cell expressing a CD7- targeting polypeptide and one or more microRNA (miRNA) that functionally down-regulates a target in the cell.

[0015] In an embodiment, the CD7-targeting polypeptide specifically binds to CD7.

[0016] In some embodiments the CD7-targeting polypeptide may be an engineered T-cell receptor targeting CD7, such as a chimeric antigen receptor (CAR) targeting CD7.

[0017] In an embodiment, the miRNA improves the expansion of the cell, reduces the alloreactivity of the cell, reduces the immune system rejection of the cell, reduces the lymphodepletion of the cell, reduces the fratricide of the cell and / or improves the potency and persistence of the cell for durable treatment outcomes.

[0018] In an embodiment, the miRNA functionally down-regulates one or more of TCR, CD7, HLA- I and HLA-II.

[0019] In a preferred embodiment of the present invention, the miRNA functionally down-regulates CD7 by inhibiting the expression of CD7.

[0020] In an embodiment, the miRNA functionally down-regulates TCR by inhibiting the expression of TCRa, TCRb, CD3d, CD3g, CD3e and / or CD3z.

[0021] In an embodiment the miRNA functionally down-regulates HLA-II by inhibiting the expression of one or more of a HLA-II polypeptide, CIITA, RFX5, RFXANK, and / or RFXAP.

[0022] In an embodiment the engineered donor cell additionally expresses an miRNA which functionally down-regulates one or more of CD5, B2M, CD3z, CD3e, CD3d, CD3g, TCRa, TCRb, CIITA, RFX5, RFXANK, RFXAP, TIM-3, PD-1, CD52, TIGIT, LAG-3, CTLA-4, DNMT3A, NR4A3, PRDM1, TGFBR2, REGNASE-1, ROQUIN-1, CCR5 and GM-CSF by inhibiting the expression thereof.

[0023] In one embodiment, one or more surface-expressed polypeptides involved in immune signalling is up-regulated, optionally by a transcript expressing the polypeptide. In an embodiment, the up-regulated surface-expressed polypeptide involved in immune signalling may be a non-classical HLA class I polypeptide, CD47 and / or PD-L1.

[0024] In an embodiment a surface expressed non-classical HLA class I is up-regulated and a surface- expressed HLA class I is down-regulated. In an embodiment, the non-classical HLA class I polypeptide is a genetically modified HLA-E, HLA-G, or HLA-F polypeptide.

[0025] In an embodiment of the present invention, a surface expressed CAR is up-regulated and a surface expressed TCR is down-regulated.

[0026] In an embodiment of the present invention, the engineered donor cell expresses a safety switch gene or suicide gene.

[0027] In a second aspect, the invention provides a nucleic acid construct comprising a sequence encoding a CD7-targeting polypeptide and one or more miRNA hairpins.

[0028] In an embodiment of the nucleic acid construct of the present invention, the nucleic acid construct comprises at least a first and a second miRNA hairpin, wherein the first miRNA hairpin and the second miRNA hairpin target a combination of two sequences independently selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 1 to 128 or 282-293.

[0029] In an embodiment the CD7-targeting polypeptide comprises a polypeptide encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134.

[0030] In another embodiment the CD7-targeting polypeptide comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

[0031] In an embodiment of the nucleic acid construct of the present invention, the construct comprises at least two different miRNA hairpins which target different regions of the same transcript.

[0032] In an embodiment, the construct comprises at least two different miRNA hairpins which target different transcripts of the same gene. In an embodiment, the construct comprises at least two different miRNA hairpins which target different splice variants of the same gene.

[0033] In an embodiment there are two copies of the first miRNA hairpin and / or two copies of the second miRNA hairpin. In an embodiment there are three copies of the first miRNA hairpin and / or two copies of the second miRNA hairpin.

[0034] In an embodiment of the nucleic acid construct of the present invention, the construct comprises a promoter element configured to express the miRNA hairpin(s), optionally wherein the promoter element is a promoter.

[0035] In an embodiment, the promoter is a eukaryotic promoter, optionally wherein the eukaryotic promoter is a Pol II or Pol III promoter.

[0036] In an embodiment, the promoter is an inducible promoter, a tissue-specific promoter, a cell lineage-specific promoter or a synthetic promoter.

[0037] In an embodiment, the promoter is a UBI promoter. In an embodiment, the promoter is an EFla promoter, a derivative of an EFla promoter or an EFl short promoter, preferably the promoter is an EFl short promoter.

[0038] In an embodiment of the nucleic acid construct of the present invention, the nucleic acid construct further comprises a promoter element configured to express the CD7-targeting polypeptide, optionally wherein the promoter element is a promoter. In an embodiment, the promoter configured to express the CD7-targeting polypeptide is a PGK promoter, an EFla promoter, a derivative of an EFla promoter or an EFl short promoter.

[0039] In an embodiment of the nucleic acid construct of the present invention, the nucleic acid construct further comprises a spacer, optionally wherein the spacer comprises an enhancer, further optionally wherein the spacer is an enhancer.

[0040] In an embodiment, the spacer is at least 50 nucleotides in length.

[0041] In an embodiment, the spacer is between 50 and 1,000 nucleotides in length. In an embodiment, the spacer is between 50 and 900, 50 and 800, 100 and 800, or 50 and 800 nucleotides in length.

[0042] In an embodiment, the spacer is at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 200 nucleotides in length. In an embodiment, the spacer is a GFP sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 141.

[0043] In an embodiment, the spacer is positioned between the promoter configured to express the miRNA hairpins and the miRNA hairpins.

[0044] In an embodiment, the spacer is heterologous with respect to the promoter element. In an embodiment, the spacer comprises an encoded open reading frame.

[0045] In an embodiment of the nucleic acid construct of the present invention, the CD7-targeting polypeptide specifically binds to CD7, optionally wherein the CD7-targeting polypeptide is an engineered T-cell receptor targeting CD7, optionally a CAR targeting CD7. In an embodiment, the sequence encoding a CD7-targeting polypeptide comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134, and / or wherein the CD7-targeting polypeptide comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

[0046] In an embodiment of the nucleic acid construct of the present invention, the miRNA hairpins are under the control of a first promoter and the sequence encoding a CD7-targeting polypeptide is under the control of a second promoter.

[0047] In another embodiment the miRNA hairpins and the sequence encoding a CD7-targeting polypeptide are under the control of a single promoter.

[0048] In an embodiment of the nucleic acid construct of the present invention, the construct further comprises a selection gene.

[0049] In an embodiment, the selection gene is LNGFR, truncated endothelial growth factor receptor (tEGFR), tCD19, CD20 or a truncated CD20 (tCD20), tCD34 or a derivative thereof.

[0050] In an embodiment of the nucleic acid construct of the present invention, the construct further comprises a sequence encoding a suicide gene or safety switch gene. In an embodiment, the suicide gene or safety switch gene is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), inducible caspase 9 (iCasp9), truncated endothelial growth factor receptor (tEGFR), RQR8, dihydrofolate reductase (DHFR), CD20 or a truncated CD20 (tCD20), thymidylate synthase (TYMS) and lenalidomide OFF-switch CAR molecules.

[0051] In an embodiment of the nucleic acid construct of the present invention, the construct further comprises an internal ribosome entry site (IRES).

[0052] In a third aspect, the invention provides a DNA molecule, plasmid or vector comprising the nucleic acid construct of the present invention, optionally wherein the vector is an expression vector, optionally wherein the expression vector is an adenovirus, an adeno-associated virus, a retrovirus or lentivirus vector.

[0053] In a fourth aspect, the invention provides a method for preparing an engineered donor cell comprising transfecting or transducing a cell with the nucleic acid construct, DNA molecule, plasmid or vector of the present invention.

[0054] In an embodiment, the invention provides a method for preparing an engineered donor cell from a patient donor or healthy donor comprising: (a) collecting a cell from the donor; and (b) transfecting or transducing the cell with the nucleic acid construct, DNA molecule, plasmid or vector of the present invention; and (c) expressing the nucleic acid construct. In an embodiment, the engineered donor cell is a T-cell.

[0055] It will be understood that in some embodiments the invention encompasses in vivo delivery of the nucleic acid construct, DNA molecule, plasmid or vector of the present invention to a cell.

[0056] In an embodiment, the nucleic acid construct, DNA molecule, plasmid or vector down- regulates a TCR polypeptide and up-regulates a CAR polypeptide targeting CD7. The engineered donor cell may be a CAR T-cell.

[0057] In a fifth aspect, the invention provides an engineered donor cell obtainable or obtained by the methods of the present invention.

[0058] In a sixth aspect, the invention provides a cell comprising the nucleic acid construct, DNA molecule, plasmid or vector of the present invention, optionally wherein the cell is an engineered donor cell. In an embodiment of the engineered donor cell of the invention, the engineered donor cell is a eukaryotic cell, such as a mammalian cell.

[0059] In an embodiment, the engineered donor cell in an immune effector cell.

[0060] In an embodiment, the immune effector cell is selected from the group comprising: alpha-beta T-cells, gamma-delta T-cells, tumour infiltrating lymphocytes (TILS), TCR-engineered T- cells, CAR T-cells, NK cells, NK / T-cells, T regulatory cells, mucosal-associated invariant T- cells (MAIT), monocytes and macrophages. In an embodiment, the immune effector cell is a CAR T-cell.

[0061] In an embodiment of the engineered donor cell of the invention, the engineered donor cell is a stem cell or a progenitor cell.

[0062] In an embodiment, the engineered donor cell is a pluripotent stem cell, such as an embryonic and / or an induced pluripotent stem cell. In an alternative embodiment, the engineered donor cell is a multipotent stem cell, such as a haematopoietic stem cell.

[0063] In an embodiment of the engineered donor cell of the invention, the engineered donor cell is a differentiated cell.

[0064] In a seventh aspect, the invention provides a composition comprising the engineered donor cell of the present invention, optionally further comprising a pharmaceutically acceptable carrier, excipient, diluent or salt.

[0065] In an eighth aspect, the invention provides an in vitro, ex vivo or in vivo method of depleting CD7+ cells, comprising contacting the CD7+ cells with the engineered donor cell or composition of the present invention.

[0066] In a ninth aspect, the invention provides the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of the present invention for use in a method of treating a disease.

[0067] In an embodiment the method of treating a disease comprises administering the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of the present invention to a subject in need thereof. In an embodiment the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of the present invention are for use in the manufacture a medicament for the treatment of a disease.

[0068] In an embodiment, the disease may be selected from cancer, an infectious disease, an autoimmune disease or an inherited disorder. In an embodiment, the cancer is a T-cell malignancy, such as leukaemia or lymphoma.

[0069] In an alternate embodiment, the disease is a CD7+ cell-related disease.

[0070] In an eleventh aspect, the invention provides a miRNA hairpin, wherein the miRNA hairpin targets a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 1 to 128 or 282-293.

[0071] In twelfth aspect, the invention provides a miRNA hairpin, wherein the miRNA hairpin comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 142-281.

[0072] In a thirteenth aspect, the invention provides a VHH antibody or antibody fragment, wherein the VHH antibody or antibody fragment targets a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

[0073] BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 A&B: Nucleic acid constructs comprising a gene silencing cassette and gene expression cassette. Shown are the six miRNA hairpins for silencing target genes, and the CD7 VHH as part of the anti-CD7 CAR, alongside a RQR8 reporter / safety-switch, separated by a 2A peptide.

[0075] Figure 2: Downregulation of CD7 expression in gene-modified primary T-cells, presented as flow cytometric histograms with normalized values of the percentage of cells expressing CD7 and MFI. The histograms are based on gating on gene modified, mCherry positive T-cells.

[0076] Figure 3: Expansion of gene-modified T-cells after transduction with the bimodal construct bearing a control anti-CD7 CAR (CAR7) construct and with / without CD7 silencing using miRNA CD7 T12. Superior expansion of the cells is achieved when CD7 silencing is incorporated, demonstrating the functional relevance of CD7 silencing.

[0077] Figure 4A: Schematic representation of the CAR T-cell manufacturing process.

[0078] Figure 4B: Expansion of the miCAR7 T-cells during manufacturing. All miCAR constructs had TCR, HLA-I and CD7 silencing, with an interchange of VHH binders on the CAR: 1185 (Fl 1), 1186 (BIO), 1187 (F04), 1188 (Cl l), 1189 (DOI), 1190 (G02). UTD, Untransduced T- cells.

[0079] Figure 4C: Immunophenotypic characterization of miCAR7 T-cells after depletion of TCR a / p positive cells, as shown in flow cytometry dot plots. All miCAR constructs had TCR, HLA- I and CD7 silencing, with an interchange of VHH binders on the CAR: 1185 (F 11), 1186 (BIO), 1187 (F04), 1188 (Cl l), 1189 (DOI), 1190 (G02). UTD, Untransduced T-cells. CAR positive cells were detected based on RQR8 co-expression (CD34). Remaining cells after TCR a / p depletion are devoid of TCR a / p, silenced for HLA-ABC and CD7, and fully express RQR8 reporter gene (CD34).

[0080] Figure 4D: Immunophenotypic characterization of miCAR7 T-cells after depletion of TCR a / p positive cells, as shown in percentages of cells and mean fluorescence intensity (MFI). All miCAR constructs had TCR, HLA-I and CD7 silencing, with an interchange of VHH binders on the CAR: 1185 (Fl 1), 1186 (BIO), 1187 (F04), 1188 (Cl l), 1189 (DOI), 1190 (G02). UTD, Untransduced T-cells. The anti-CD7 CAR was detected by means of recombinant CD7 protein (RecCD7), while RQR8 co-expression is detected by means of anti-CD34 antibody.

[0081] Figure 5: Specific cytotoxicity of tumor cells by miCAR7 T-cells. In short-term cytotoxicity assays, miCAR7 T-cells with varied anti-CD7 VHH did not perform equally well in terms of functional activity against CD7-expressing tumor cells. Only VHH BIO, Cl l, DOI and G02 proved able to efficiently eliminate CD7+ tumor cells. Engineered miCAR7 T-cells (effector, E) and SUP-T1 cells (target, T) were co-cultured at E:T ratios of 1 : 1, 1 :3 and 1 :9 over 24, 48, 72 and 96 hours. Target cell survival (SUP-T1 cells labelled with CTV) was assessed by detection of CTV positivity on flow cytometry after the indicated time points. In a long-term recursive killing assay, only miCAR7 T-cells with anti-CD7 VHH BIO, Cl l, DOI and G02 efficiently depleted MOLT-4 or SUP-T1 tumor cells either over four rounds or between two to three rounds of re-stimulation with the same number of target cells, respectively. Figure 6: Tolerability assessment of BIO VHH miCAR7 T-cells in NSG mice. Mice were dosed with miCAR7 T cells expressing the BIO VHH binder, which was previously shown to have effective on target activity in vitro. These miCAR7 T-cells also had silencing of TCRa / b, HLA-I and CD7, allowing for purification to deplete remaining TCRa / b expressing T-cells. Mice (n=3) received a single dose of 7.5xlOA6 cells via tail vein injection on Study Day 1, after which body weights were measured daily for two weeks. Relative body weight curves over the 15-day period are shown, expressed as a percentage of initial body weight. Body weight was measured daily to assess tolerability and general health of the mice. Data are presented as individual values for each mouse. No significant weight loss or adverse effects were observed during the study period, indicating that miCAR7 T-cells were well-tolerated by the NSG mice during the 15-day observation period. This outcome implies that the administered dose of 7.5 million CAR T cells did not induce severe toxicity or cachexia-like symptoms in the treated animals.

[0082] Figure 7A: Anti-leukemic activity of BIO VHH miCAR7 T cells with silencing of three target genes in an ALL xenograft mouse model. Mice were dosed with miCAR7 T cells expressing the BIO VHH binder, along with silencing of TCRa / b, HLA-I and CD7. A schematic of the experimental setup of the CCRF-CEM xenograft model. miCAR7 T cells (0.2 x 106, 1.5 x 106or 6.5 x io6) or miCAR19 T-cells (6.4 x 106) were injected 5 days after engraftment of luciferase-labelled CCRF-CEM cells (3 x 105). BLI was performed before T-cell injection on day 4 and twice-a-week thereafter (n = 6 mice per treatment group).

[0083] Figure 7B: Bioluminescence imaging (BLI) on the indicated days of mice with CCRF-CEM cell engraftment and treatment with vehicle only (PBS), miCAR7 or miCAR19 T cells.

[0084] Figure 7C: Quantitative analysis of BLI signals for individual mice from each treatment group, indicating that miCAR7 T cells demonstrate dose-dependent antitumor activity, with the highest dose effectively controlling tumor growth, while the two lower doses exhibited reduced efficacy.

[0085] Figure 8A: Immunophenotypic characterization of multiplex engineered miCAR7 T-cells with silencing of four target genes. Anti-CD7 CAR and RQR8 safety switch expression on T-cells following purification by depletion of TCRa / b positive cells. CAR molecules were detected using fluorescently labelled recombinant CD7 protein, while RQR8 co-expression was detected by means of anti-CD34 antibody. Figure 8B: Residual expression of silenced targets based on relative mean fluorescence intensity (MFI) when compared to untransduced T-cells. As expected, negligible TCRa / b expression remained following depletion, while the same was true for HLA-II (HLA- DPDQDR) and CD7 expression. Tuned HLA-I (HLA-ABC) silencing within the range of 75- 85% was obtained using the optimized gene constructs carrying B2M_T2 targeting miRNA. All miCAR constructs were designed to silence TCRa / b, HLA-I, HLA-II and CD7; with an interchange of VHH binders on the CAR: 1293 (BIO), 1549 (Cl l), 1567 (DOI), 1570 (G02).

[0086] Figure 9A: Specific cytotoxicity of tumor cells by miCAR7 T-cells with silencing of four target genes. (A) In short-term cytotoxicity assays, miCAR7 T-cells with varied anti-CD7 VHH perform equally well in terms of functional activity against CD7-expressing tumor cells. Engineered miCAR7 T-cells (effector, E) and tumor cells (target, T) were co-cultured at E:T ratios of 1: 1 over 24, 48, and 72 hours. Target cell survival (GFP-expressing MOLT-4 or SUP- T1 cells) was assessed by detection of GFP positivity on flow cytometry after the indicated time points. Anti-CD19 CAR T-cells with silencing of TCRa / b and HLA-I were used as controls.

[0087] Figure 9B: In a long-term recursive killing assay, miCAR7 T-cells were also able to efficiently deplete MOLT-4 or SUP-T1 tumor cells either between one (1549) and six (1293, 1567 and 1570) rounds or two rounds of re-stimulation with the same number of target cells, respectively. All miCAR constructs were designed to silence TCRa / b, HLA-I, HLA-II and CD7, with an interchange of VHH binders on the CAR: 1293 (BIO), 1549 (Cl l), 1567 (DOI), 1570 (G02). Anti-CD19 CAR T-cells with silencing of TCRa / b and HLA-I were used as controls.

[0088] DETAILED DESCRIPTION

[0089] The present invention relates generally to engineered donor cells, nucleic acid constructs, miRNA hairpins, VHH antibody binders, methods for preparing engineered donor cells, methods of treating using the engineered donor cell, nucleic acid construct or other products of the invention.

[0090] The present invention relates to engineered donor cells expressing CD7-targetting polypeptide and one or more microRNAs (miRNAs).

[0091] The one or more miRNAs functionally down-regulates a target in the cell. CD7 targeting polypeptide

[0092] The engineered donor cell and nucleic acid construct of the present invention express a CD7- targeting polypeptide.

[0093] The CD7-targeting polypeptide may be an engineered T-cell receptor targeting CD7, such as a chimeric antigen receptor (CAR) targeting CD7.

[0094] In a preferred embodiment the CD7-targeting polypeptide comprises a polypeptide encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134.

[0095] In a preferred embodiment the CD7-targeting polypeptide comprises a polypeptide encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134.

[0096] In another embodiment the CD7-targeting polypeptide comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

[0097] In a preferred embodiment the CD7-targeting polypeptide comprises a polypeptide encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

[0098] Table 1 - CD7 targeting nucleotide sequences

[0099] Table 2 - CD7 targeting polypeptide sequences

[0100] MicroRNAs (miRNAs)

[0101] The present invention includes one or more miRNAs. miRNAs are a form of inhibitory nucleic acids. Inhibitory nucleic acids are well known in the art. For example, siRNA, shRNA and doublestranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Publications 2003 / 0051263, 2003 / 0055020, 2004 / 0265839, 2002 / 0168707, 2003 / 0159161, and 2004 / 0064842, all of which are herein incorporated by reference in their entirety.

[0102] Since the discovery of RNA interference (RNAi) by Fire and colleagues in 1998, the biochemical mechanisms have been rapidly characterized. Double stranded RNA (dsRNA) is cleaved by Dicer, which is an RNAase III family ribonuclease. This process yields miRNAs of ~21 nucleotides in length. These miRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC) that is guided to target mRNA. RISC cleaves the target mRNA in the middle of the complementary region. In mammalian cells, the related miRNAs are found that are short RNA fragments (~22 nucleotides). miRNAs are generated after Dicer-mediated cleavage of longer (~70 nucleotide) precursors with imperfect hairpin RNA structures. The miRNA is incorporated into a miRNA-protein complex (miRNP), which leads to translational repression of target mRNA.

[0103] In designing RNAi molecules there are several factors that may considered such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system. Furthermore, the RNAi process is homology dependent, so the sequences are often carefully selected so as to maximize gene specificity, while minimizing the possibility of crossinterference between homologous, but not gene-specific sequences. Particularly, the miRNA often exhibits greater than 80, 85, 90, 95, 98% or even 100% identity between the sequence of the miRNA and a portion of the nucleotide sequence of a target gene. Sequences less than about 80% identical to the target gene may be substantially less effective. Thus, the greater identity between the miRNA and the target gene to be inhibited, the less likely expression of unrelated genes will be affected.

[0104] In addition, the size of the miRNA is an important consideration. In some embodiments of the present invention, the miRNA may be at least about 19-25 nucleotides. In the context of the present invention, the miRNA is particularly less than 500, 200, 100, 50, 25, 24, 23 or 22 nucleotides in length. In some embodiments, the miRNA is from about 15 nucleotides to about 25 nucleotides or from about 19 nucleotides to about 25 nucleotides in length. In some embodiments the miRNA may be 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 12, 11 or 10 nucleotides in length. In some embodiments the miRNA may be 19 nucleotides in length. In some embodiments the miRNA may be 20 nucleotides in length. In some embodiments the miRNA may be 21 nucleotides in length. In some embodiments the miRNA may be 22 nucleotides in length.

[0105] To improve the effectiveness of miRNA-mediated gene silencing, guidelines for selection of target sites on mRNA have been developed for optimal design of miRNA (Soutschek et al., 2004; Wadhwa et al., 2004). These strategies may allow for rational approaches for selecting siRNA sequences to achieve maximal gene knockdown. To facilitate the entry of miRNA into cells and tissues, a variety of vectors including plasmids and viral vectors such as adenovirus, lentivirus, and retrovirus have been used (Wadhwa et al., 2004).

[0106] Typically, an inhibitory nucleic acid forms a double-stranded structure; the double-stranded structure may result from two separate nucleic acids that are partially or completely complementary.

[0107] In certain embodiments of the present invention, the miRNA may comprise only a single nucleic acid (polynucleotide) or nucleic acid analogue and form a double- stranded structure by complementing with itself (e.g., forming a hairpin loop). The double-stranded structure of the miRNA may comprise 16 - 500 or more contiguous nucleobases, including all ranges therebetween. The miRNA may comprise 17 to 35 contiguous nucleobases, more particularly 18 to 30 contiguous nucleobases, more particularly 19 to 25 nucleobases, more particularly 20 to 23 contiguous nucleobases, or 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure. It is therefore the case that miRNA may also be described as miRNA-embedded shRNA. miRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of a number of techniques well-known to those of ordinary skill in the art. For example, commercial sources of predesigned miRNA include Invitrogen’s StealthTM Select technology (Carlsbad, CA), Ambion® (Austin, TX), and Qiagen® (Valencia, CA).

[0108] In a most preferred embodiment, the miRNAs included within the present invention are constructed according to WO2019186274, in respect of the miRNA architecture and design described therein, which is incorporated by reference herein in its entirety. The nucleic acid construct of the invention comprises a miRNA which functionally down- regulates a target in the cell.

[0109] Herein the miRNA is preferably derived from a miRNA hairpin.

[0110] In one embodiment the miRNA improves the expansion of the cell, reduces the alloreactivity of the cell, reduces the immune system rejection of the cell, reduces the lymphodepletion of the cell, reduces the fratricide of the cell and / or improves the potency and persistence of the cell for durable treatment outcomes.

[0111] In one embodiment the miRNA functionally down-regulates one or more of TCR, CD7, HLA- I and HLA-II by inhibiting the expression thereof.

[0112] In a preferred embodiment of the present invention, the miRNA functionally down-regulates CD7 by inhibiting the expression of CD7.

[0113] In an embodiment, the miRNA functionally down-regulates TCR such as by inhibiting the expression of TCRa, TCRb, CD3d, CD3g, CD3e and / or CD3z.

[0114] In an embodiment the miRNA functionally down-regulates HLA-II such as by inhibiting the expression of one or more of a HLA II polypeptide, CIITA, RFX5, RFXANK, and / or RFXAP.

[0115] In one embodiment the nucleic acid construct comprises a miRNA which functionally downregulates TCR and a miRNA which functionally downregulates CD7.

[0116] In one embodiment the nucleic acid construct comprises a miRNA which functionally downregulates TCR and a miRNA which functionally downregulates HLA-I.

[0117] In one embodiment the nucleic acid construct comprises a miRNA which functionally downregulates TCR and a miRNA which functionally downregulates HLA-II.

[0118] In one embodiment the nucleic acid construct comprises a miRNA which functionally downregulates CD7 and a miRNA which functionally downregulates HLA-I.

[0119] In one embodiment the nucleic acid construct comprises a miRNA which functionally downregulates CD7 and a miRNA which functionally downregulates HLA-II.

[0120] In one embodiment the nucleic acid construct comprises a miRNA which functionally downregulates HLA I and a miRNA which functionally downregulates HLA-II. In one embodiment the nucleic acid construct of the invention comprises miRNAs which functionally down-regulates two, three, four, five, six, seven, eight, nine, ten or more targets.

[0121] In one embodiment the nucleic acid construct of the invention comprises one, two, three, four, five, six, seven, eight, nine or ten miRNAs.

[0122] In one embodiment the nucleic acid construct may comprise two copies of a first miRNA hairpin and two copies of a second miRNA hairpin.

[0123] The another embodiment the nucleic acid construct may comprise three copies of a first miRNA hairpin and three copies of a second miRNA hairpin.

[0124] The another embodiment the nucleic acid construct may comprise at least two different miRNA hairpins which target different regions of the same transcript.

[0125] The another embodiment the nucleic acid construct may comprise at least two different miRNA hairpins which target different transcripts of the same gene.

[0126] The another embodiment the nucleic acid construct may comprise at least two different miRNA hairpins which target different splice variants of the same gene.

[0127] In an embodiment the engineered donor cell additionally expresses an miRNA which functionally down-regulates one or more of CD5, B2M, CD3z, CD3e, CD3d, CD3g, TCRa, TCRb, CIITA, RFX5, RFXANK, RFXAP, TIM-3, PD-1, CD52, TIGIT, LAG-3, CTLA-4, DNMT3A, NR4A3, PRDM1, TGFBR2, REGNASE-1, ROQUIN-1, CCR5 and GM-CSF by inhibiting the expression thereof.

[0128] In one embodiment, the miRNA may target one or more of the sequences included in Table 3, below.

[0129] Table 3: Target sequences for miRNAs

[0130] In Table 3 above, the first column indicates the target protein (for ease of identification, some mere alternative designations of the targets are also indicated); the second column indicates the identifier of the target sequence, and the fourth column indicates the sequence targeted by the miRNA. Thus, it is understood for example that, for any miRNA of the present invention which includes a miRNA hairpin targeting CD7, the miRNA hairpin targeting CD7 can preferably target any of CD7 1 to CD7 12, which are defined by the sequences provided in the fourth column. This applies for every gene listed in Table 3 and the associated target sequences.

[0131] In an embodiment, the miRNA included within the engineered donor cell or nucleic acid construct of the present invention targets a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence listed in Table 3.

[0132] In an embodiment, the miRNA included within the engineered donor cell or nucleic acid construct of the present invention targets a sequence having one, two, three, four or five mismatches to a sequence listed in Table 3.

[0133] In a preferred embodiment the miRNA targets a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-128 or 282-293.

[0134] In a preferred embodiment the miRNA targets a polynucleotide sequence comprising or consisting of any one of SEQ ID NO: 1-128 or 282-293.

[0135] In an embodiment, the miRNA targets a sequence having one, two, three, four or five mismatches to any one of SEQ ID NO: 1-128 or 282-293.

[0136] Table 4: miRNA hairpin sequences

[0137] In Table 4 above, the first column indicates the target protein (for ease of identification, some mere alternative designations of the targets are also indicated); the second column indicates the identifier of the miRNA hairpin sequence, and the fourth column indicates the sequence of the miRNA hairpin. Thus, it is understood for example that, for any miRNA of the present invention which includes a miRNA hairpin targeting CD7, the miRNA hairpin targeting CD7 can may be any of CD7 1 to CD7 12, which are defined by the sequences provided in the fourth column. This applies for every gene listed in Table 4 and the associated miRNA hairpin sequences.

[0138] In an embodiment, the miRNA hairpin included within the engineered donor cell or nucleic acid construct of the present invention has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a miRNA hairpin sequence listed in Table 4.

[0139] In an embodiment, the miRNA hairpin included within the engineered donor cell or nucleic acid construct of the present invention may have one, two, three, four or five mutations relative to a sequence listed in Table 4.

[0140] In an embodiment the invention also contemplates the use of truncated versions of the miRNA hairpin sequences. Such truncated versions may be lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 20, 30, 35, 40, 45, 50 or more nucleotides from either of both ends of the sequences recited in Table 4.

[0141] In a preferred embodiment the miRNA hairpin included within the engineered donor cell or nucleic acid construct of the present invention has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 142-281.

[0142] In a preferred embodiment the miRNA hairpin sequence comprises or consists of any one of SEQ ID NO: 142-281.

[0143] In an embodiment, the miRNA hairpin included within the engineered donor cell or nucleic acid construct of the present invention comprises or consists of any one of SEQ ID NO: 142- 281. The miRNAs and miRNA hairpins described above fall within the scope of the invention, such that the invention includes the miRNAs and miRNA hairpins independent on an engineered donor cell or a nucleic acid construct.

[0144] For example, the invention includes an miRNA, wherein the miRNA hairpin targets a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence listed in Table 3.

[0145] In an embodiment, the miRNA targets a sequence having one, two, three, four or five mismatches to a sequence listed in Table 3.

[0146] In a preferred embodiment the miRNA targets a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-128 or 282-293.

[0147] In a preferred embodiment the miRNA targets a polynucleotide sequence comprising or consisting of any one of SEQ ID NO: 1-128 or 282-293.

[0148] In an embodiment, the miRNA targets a sequence having one, two, three, four or five mismatches to any one of SEQ ID NO: 1-128 or 282-293.

[0149] In an embodiment, the miRNA hairpin has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a miRNA hairpin sequence listed in Table 4.

[0150] In an embodiment, the miRNA hairpin may have one, two, three, four or five mutations relative to a sequence listed in Table 4.

[0151] In an embodiment the invention also contemplates truncated versions of the miRNA hairpin sequences. Such truncated versions may be lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 11, 12, 13, 14, 15, 20, 20, 30, 35, 40, 45, 50 or more nucleotides from either of both ends of the sequences recited in Table 4.

[0152] In a preferred embodiment the miRNA hairpin has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 142-281.

[0153] In a preferred embodiment the miRNA hairpin sequence comprises or consists of any one of SEQ ID NO: 142-281. In an embodiment, the miRNA hairpin comprises or consists of any one of SEQ ID NO: 142- 281.

[0154] Upregulation of surface-expressed polypeptides

[0155] In some embodiments of the invention, one or more surface-expressed polypeptides of the engineered donor cell may be upregulated.

[0156] In some embodiments the one or more surface-expressed polypeptides may be upregulated by a transcript expressing the polypeptide.

[0157] In some embodiments the one or more surface-expressed polypeptides may be involved in immune signalling.

[0158] In some embodiments the up-regulated surface-expressed polypeptide involved in immune signalling is a non-classical HLA class I polypeptide, CD47 and / or PD-L1.

[0159] In some embodiments a surface expressed non-classical HLA class I is up-regulated and a surface-expressed HLA class I is down-regulated.

[0160] In some embodiments the surface-expressed HLA class I may be down-regulated by a miRNA.

[0161] In some embodiments the non-classical HLA class I polypeptide is a genetically modified HLA-E, HLA-G, or HLA-F polypeptide.

[0162] In some embodiments a surface expressed CAR is up-regulated and a surface expressed TCR is down-regulated.

[0163] In some embodiments the surface-expressed CAR may be down-regulated by a miRNA.

[0164] Engineered donor cell

[0165] The engineered donor cell may be derived from any cell.

[0166] In some embodiments the engineered donor cell is a eukaryotic cell, such as a mammalian cell.

[0167] In some embodiments the engineered donor cell in an immune effector cell. In some embodiments the immune effector cell is selected from the group comprising: alphabeta T-cells, gamma-delta T-cells, tumour infiltrating lymphocytes (TILS), TCR-engineered T-cells, CAR T-cells, NK cells, NK / T cells, T regulatory cells, mucosal-associated invariant T-cells (MAIT), monocytes and macrophages.

[0168] In some embodiments the engineered donor cell is a T-cell.

[0169] In some embodiments the immune effector cell is a CAR T-cell.

[0170] In some embodiments the engineered donor cell is a stem cell or a progenitor cell.

[0171] In some embodiments the engineered donor cell is a pluripotent stem cell, such as an embryonic and / or an induced pluripotent stem cell.

[0172] In some embodiments the engineered donor cell is a multipotent stem cell, such as a haematopoietic stem cell

[0173] In some embodiments the engineered donor cell may be derived from a subject, such as a human subject.

[0174] In some embodiments the human subject may be a healthy human subject.

[0175] In some embodiments the human subject may be suffering from a disease. In this case the “subject” may be referred to as a “patient”.

[0176] Antibodies

[0177] The invention encompasses an antibody which targets a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the CD7 targeting polypeptides disclosed herein.

[0178] In one embodiment the antibody which targets a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 135-140.

[0179] In one embodiment the antibody targets a polypeptide sequence comprising or consisting of any one of SEQ ID NOs: 135-140.

[0180] In one embodiment the antibody is a VHH or VHH fragment. Nucleic acid construct

[0181] Within the invention expression vectors may be employed to express a nucleic acid construct of the invention. Expression requires that appropriate signals be provided in the vectors, which include various regulatory elements, such as enhancers / promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize RNA stability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.

[0182] Regulatory Elements

[0183] Throughout this application, the term “expression construct” or “expression vector” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest i.e., as is the case with RNA molecules of the embodiments.

[0184] In certain embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter element, which may be a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

[0185] The term “promoter” will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for eukaryotic RNA polymerase (Pol) I, II or III. Much of the thinking about how promoters are organized derives from analyses of several viral Pol II promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxy nucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0186] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0187] In some embodiments, the promoter comprises an Elongation Factor 1 short (EFIs) promoter. In other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest.

[0188] In some embodiments of the invention the nucleic acid construct comprises a promoter element configured to express the miRNA hairpins. The promoter element may be a promoter.

[0189] In some embodiments the promoter configured to express the miRNA hairpins may be a eukaryotic promoter, such as a Pol II or Pol III promoter.

[0190] In some embodiments the promoter configured to express the miRNA hairpins may be an inducible promoter, a tissue-specific promoter, a cell lineage-specific promoter or a synthetic promoter.

[0191] In some embodiments the promoter configured to express the miRNA hairpins may be a UBI promoter.

[0192] In some embodiments the promoter configured to express the miRNA hairpins is an EFla promoter, a derivative of an EFla promoter or an EFl short promoter. The promoter configured to express the miRNA hairpins is preferably an EFl short promoter.

[0193] In some embodiments of the invention the nucleic acid construct comprises a promoter element configured to express the CD7-targeting polypeptide. The promoter element may be a promoter.

[0194] In some embodiments the promoter configured to express the CD7-targeting polypeptide may be a PGK promoter, an EFla promoter, a derivative of an EFla promoter or an EFl short promoter.

[0195] In some embodiments the miRNA and the CD7-targeting polypeptide may be under the control of a single promoter.

[0196] In some embodiments the miRNA and the CD7-targeting polypeptide may be under the control of individual promoters. Herein the individual promoters are independently selected such that they may be the same or different types of promoters.

[0197] In embodiments wherein the nucleic acid construct comprises more than one miRNA, the miRNAs may be under the control of a single promoter or under the control of an individual promoter per miRNA. Herein the individual promoters are independently selected such that they may be the same or different types of promoters.

[0198] In another embodiment wherein the nucleic acid construct comprises more than one miRNA, the miRNAs may be divided such that some miRNAs are under the control of one promoter and some miRNAs are under the control of another promoter. For example, if a nucleic acid construct contains six miRNAs, four miRNAs may be under the control of one promoter and two miRNAs may be under the control of a second promoter or three miRNAs may be under the control of one promoter and three miRNAs may be under the control of a second promoter.

[0199] The use of other viral or mammalian cellular or bacterial phage promoters which are well- known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for the given purpose.

[0200] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

[0201] Below is a list of viral promoters, cellular promoters / enhancers and inducible promoter s / enhancers that can be used in relation to the invention (Table 5 and Table 6). Additionally, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the CD7 binding polypeptide and / or miRNA.

[0202] Truncated promoters may also be used to drive expression. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0203] Spacer

[0204] In some embodiments the nucleic acid construct of the invention may comprise a spacer. The presence of a spacer appears to enhance knockdown efficiency of miRNA (Stegmeier et al., 2005). Spacers may be any nucleotide sequence.

[0205] In some embodiments the spacer may be at least 50 nucleotides in length.

[0206] In other embodiments the spacer may be between 50 and 1,000 nucleotides in length. In other embodiments the spacer may be between 50 and 900, 50 and 800, 100 and 800, or 50 and 800 nucleotides in length.

[0207] In other embodiments wherein the spacer is at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 200 nucleotides in length.

[0208] In some embodiments the spacer is a GFP derived spacer.

[0209] In a preferred embodiment the spacer has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 141.

[0210] In some embodiments the spacer comprises or consists of SEQ ID NO: 141.

[0211] Also contemplated as an element of the nucleic acid construct is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0212] Selection gene

[0213] In certain embodiments of the invention, nucleic acid construct may include a selection gene.

[0214] Such selection genes may confer an identifiable change to a cell expressing the nucleic acid construct, permitting easy identification of such cells.

[0215] Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art. In an embodiment, the selectable marker is RQR8, tEGFR or CD20.

[0216] In some embodiments the selection gene is LNGFR, truncated endothelial growth factor receptor (tEGFR), tCD19, CD20 or a truncated CD20 (tCD20), tCD34 or a derivative thereof.

[0217] Suicide gene or safety switch

[0218] In some embodiments the nucleic acid construct of the invention may comprise a suicide gene or safety switch. Herein the terms “suicide gene” and “safety switch” may be used interchangeable.

[0219] These terms refer to genes which, when activated, cause the death of a cell, usually through apoptosis or directed cytoxicity of the adaptive immune response.

[0220] In some embodiments the suicide gene or safety switch gene is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), inducible caspase 9 (iCasp9), truncated endothelial growth factor receptor (tEGFR), RQR8, dihydrofolate reductase (DHFR), CD20 or a truncated CD20 (tCD20), thymidylate synthase (TYMS) and lenalidomide OFF- switch CAR molecules.

[0221] Internal ribosome entry site (IRES)

[0222] In some embodiments the nucleic acid construct may comprise an internal ribosome entry site (IRES) to facilitate protein co-expression.

[0223] In an embodiment, the construct further comprises a peptide cleavage site. In an embodiment, the peptide cleavage site is a 2A peptide. In an embodiment, the 2A peptide is selected from the group comprising: 2A, P2A, T2A, E2A, F2A, BmCPV 2A, and BmIFV 2A.

[0224] Delivery of nucleic acid molecules and expression vectors

[0225] Within the invention, delivery of the nucleic acid construct to cells is contemplated. For example, delivery of the nucleic acid construct to a cell is contemplated in order to produce an engineered donor cell according to the invention. In certain aspects, vectors for delivery of nucleic acid constructs of the invention could be constructed to express these constructs in cells.

[0226] The invention includes a DNA molecule, plasmid or vector comprising the nucleic acid construct of the invention.

[0227] In some embodiments, the vector is an expression vector.

[0228] In some embodiments the expression vector is an adenovirus, an adeno-associated virus, a retrovirus or lentivirus vector.

[0229] In an embodiment, expressing the nucleic acid construct of the invention in a cell comprises transfecting the cell with a transposon comprising the nucleic acid construct, plasmid or vector, optionally wherein the transposon is sleeping beauty, piggyBAC or Tol2.

[0230] In some embodiments nucleic acid construct, plasmid or vector of the invention may be delivered to a cell in the form of a lipid nanoparticles (LNP). Herein, any form of LNP is envisaged.

[0231] Within the invention the use of homology directed recombination (HDR) by means of CRISPR, TALE nucleases, zinc finger nucleases, prime editing (PASSIGE) and gene writing techniques is contemplated.

[0232] In an embodiment, the cell is a human cell.

[0233] In an embodiment, the method is an in vivo method. In an embodiment, the method is an in vitro method. In an embodiment, the method is an ex vivo method.

[0234] In an embodiment, the method further comprises transplanting the cell into an organism.

[0235] In an embodiment of the method, the cell is comprised in an organism.

[0236] In particular embodiments, the following systems and methods may be used in delivery of nucleic acids to desired cell types.

[0237] Homologous recombination

[0238] In embodiments of the invention, vectors encoding nucleic acid constructs of the invention may be introduced into cells in a specific manner, for example, via homologous recombination. Current approaches to express genes in stem cells have involved the use of viral vectors (e.g., lentiviral vectors) or transgenes that integrate randomly in the genome. These approaches have not been successful due in part because the randomly integrated vectors can activate or suppress endogenous gene expression, and / or the silencing of transgene expression. The problems associated with random integration could be partially overcome by homologous recombination to a specific locus in the target genome.

[0239] Homologous recombination (HR), also known as general recombination, is a type of genetic recombination used in all forms of life in which nucleotide sequences are exchanged between two similar or identical strands of DNA. The technique has been the standard method for genome engineering in mammalian cells since the mid-1980s. The process involves several steps of physical breaking and the eventual re-joining of DNA. This process is most widely used in nature to repair potentially lethal double-strand breaks in DNA. In addition, homologous recombination produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make germ cells like sperm and ova. These new combinations of DNA represent genetic variation in offspring which allow populations to evolutionarily adapt to changing environmental conditions over time. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses. Homologous recombination is also used as a technique in molecular biology for introducing genetic changes into target organisms.

[0240] Homologous recombination can be used as targeted genome modification. The efficiency of standard HR in mammalian cells is only 10-6 to 10-9 of cells treated (Capecchi, 1990). The use of meganucleases, or homing endonucleases, such as I-Scel have been used to increase the efficiency of HR. Both natural meganucleases as well as engineered meganucleases with modified targeting specificities have been utilized to increase HR efficiency (Pingoud and Silva, 2007; Chevalier et al., 2002). Another path toward increasing the efficiency of HR has been to engineer chimeric endonucleases with programmable DNA specificity domains (Silva et al., 2011). Zinc-finger nucleases (ZFN) are one example of such a chimeric molecule in which Zinc-finger DNA binding domains are fused with the catalytic domain of a Type IIS restriction endonuclease such as FokI (as reviewed in Durai et al., 2005;

[0241] PCT / US2004 / 030606). Another class of such specificity molecules includes Transcription Activator Like Effector (TALE) DNA binding domains fused to the catalytic domain of a Type IIS restriction endonuclease such as FokI (Miller et al., 2011 : PCT / IB2010 / 000154). Also contemplated within the present invention is the use of gene editing technology Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

[0242] Nucleic acid delivery systems

[0243] One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference). Vectors include but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukaemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV etc.), adenoviral (Ad) vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors.

[0244] Episomal Vectors

[0245] The use of plasmid- or liposome-based extra-chromosomal (i.e., episomal) vectors may be also provided within the invention. Such episomal vectors may include, e.g., oriP -based vectors, and / or vectors encoding a derivative of EBV-protein EBNA-1. These vectors may permit large fragments of DNA to be introduced to a cell and maintained extra-chromosomally, replicated once per cell cycle, partitioned to daughter cells efficiently, and elicit substantially no immune response.

[0246] In particular, EBNA-1, the only viral protein required for the replication of the oriP-based expression vector, does not elicit a cellular immune response because it has developed an efficient mechanism to bypass the processing required for presentation of its antigens on MHC class I molecules (Levitskaya et al., 1997). Further, EBNA-1 can act in trans to enhance expression of the cloned gene, inducing expression of a cloned gene up to 100-fold in some cell lines (Langle-Rouault et al., 1998; Evans et al., 1997). Finally, the manufacture of such oriP-based expression vectors is inexpensive.

[0247] Other extra-chromosomal vectors include other lymphotrophic herpes virus-based vectors. Lymphotrophic herpes virus is a herpes virus that replicates in a lymphoblast (e.g., a human B lymphoblast) and becomes a plasmid for a part of its natural life-cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpes virus. Exemplary lymphotrophic herpes viruses include, but are not limited to EBV, Kaposi's sarcoma herpes virus (KSHV); Herpes virus saimiri (HS) and Marek's disease virus (MDV). Also other sources of episome-based vectors are contemplated, such as yeast ARS, adenovirus, SV40, or BPV.

[0248] One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0249] Vectors can also comprise other components or functionalities that further modulate gene delivery and / or gene expression, or that otherwise provide beneficial properties to the targeted cells. Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.

[0250] Such components also might include markers, such as detectable and / or selection markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. A large variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell's nucleus or cytoplasm.

[0251] Transposon-based system

[0252] According to a particular embodiment of the invention, the introduction of nucleic acids may use a transposon - transposase system. The used transposon - transposase system could be the well-known Sleeping Beauty, the Frog Prince transposon - transposase system (for the description of the latter see e.g., EPl 507865), or the TTAA-specific transposon piggyback system.

[0253] Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons were also once called jumping genes, and are examples of mobile genetic elements.

[0254] There are a variety of mobile genetic elements, and they can be grouped based on their mechanism of transposition. Class I mobile genetic elements, or retrotransposons, copy themselves by first being transcribed to RNA, then reverse transcribed back to DNA by reverse transcriptase, and then being inserted at another position in the genome. Class II mobile genetic elements move directly from one position to another using a transposase to "cut and paste" them within the genome.

[0255] Viral Vectors

[0256] In generating recombinant viral vectors, non-essential genes are typically replaced with a gene or coding sequence for a heterologous (or non-native) protein or nucleic acid. Viral vectors are a kind of expression construct that utilizes viral sequences to introduce nucleic acid and possibly proteins into a cell. The ability of certain viruses to infect cells or enter cells via pH- dependent or pH-independent mechanisms, to integrate their genetic cargo into a host cell genome and to express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of certain aspects of the present invention are described below.

[0257] Retroviruses have promise as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell lines (Miller, 1992).

[0258] In order to construct a retroviral vector, a nucleic acid is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g., by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid (i.e., the vector genome) to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Depending on the tropism of the envelope protein used to cover the vector particles surface, retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[0259] Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; Giry-Laterriere et al., 2011; U.S. Patents 6,013,516 and 5,994,136).

[0260] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Patent 5,994,136, incorporated herein by reference. Nucleic acid Delivery

[0261] Introduction of a nucleic acid construct of the invention may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson et al., 1989, Nabel et al., 1989), by injection (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibres (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each incorporated herein by reference); by desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985), and any combination of such methods. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

[0262] Liposome Mediated Transfection

[0263] In a certain embodiment of the invention, a nucleic acid construct of the invention may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is a nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen). The amount of liposomes used may vary upon the nature of the liposome as well as the cell used, for example, about 5 to about 20 pg vector DNA per 1 to 10 million of cells may be contemplated.

[0264] Liposome mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).

[0265] In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome encapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non histone chromosomal proteins (HMG 1) (Kato et al., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG 1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.

[0266] Electroporation

[0267] In certain embodiments of the present invention, a nucleic acid construct is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high voltage electric discharge. Recipient cells can be made more susceptible to transformation by mechanical wounding. Also the amount of vectors used may vary upon the nature of the cells used, for example, about 5 to about 20 pg vector DNA per 1 to 10 million of cells may be contemplated.

[0268] Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre B lymphocytes have been transfected with human kappa immunoglobulin genes (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur Kaspa et al., 1986) in this manner.

[0269] Calcium Phosphate

[0270] In other embodiments of the present invention, a nucleic acid construct is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse Cl 27, CHO, CV 1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).

[0271] DEAE Dextran

[0272] In another embodiment, a nucleic acid construct is delivered into a cell using DEAE dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

[0273] Cell culturing

[0274] Generally cells, such as engineered donor cells of the present invention are cultured in a culture medium, which is a nutrient-rich buffered solution capable of sustaining cell growth.

[0275] Culture media suitable for isolating, expanding and differentiating stem cells according to the method described herein include but not limited to high glucose Dulbecco's Modified Eagle's Medium (DMEM), DMEM / F-12, Liebovitz L-15, RPMI 1640, Iscove's modified Dubelcco's media (IMDM), and Opti-MEM SFM (Invitrogen Inc.). Chemically Defined Medium comprises a minimum essential medium such as Iscove's Modified Dulbecco's Medium (IMDM) (Gibco), supplemented with human serum albumin, human Ex Cyte lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine and a mitogen is also suitable. As used herein, a mitogen refers to an agent that stimulates cell division of a cell. An agent can be a chemical, usually some form of a protein that encourages a cell to commence cell division, triggering mitosis. In one embodiment, serum free media such as those described in U.S. Ser. No. 08 / 464,599 and WO96 / 39487, and the "complete media" as described in U.S. Pat. No. 5,486,359 are contemplated for use with the method described herein. In some embodiments, the culture medium is supplemented with 10% Fetal Bovine Serum (FBS), human autologous serum, human AB serum or platelet rich plasma supplemented with heparin (2U / ml). Cell cultures may be maintained in a CO2 atmosphere, e.g., 5% to 12%, to maintain pH of the culture fluid, incubated at 37°C in a humid atmosphere and passaged to maintain a confluence below 85%.

[0276] Methods for preparing an engineered donor cell The invention contemplates a method for preparing an engineered donor cell comprising transfecting or transducing a cell with the nucleic acid construct of the invention.

[0277] In some embodiments the nucleic acid construct may be presented as a DNA molecule, plasmid or vector of the invention.

[0278] In invention also provides a method for preparing an engineered donor cell from a patient donor or healthy donor comprising:

[0279] (a) collecting a cell from the donor;

[0280] (b) transfecting or transducing the cell with the nucleic acid construct, DNA molecule, plasmid or vector of the invention; and

[0281] (c) expressing the nucleic acid construct.

[0282] The cell which is to be transfected or transduced may be any cell.

[0283] In some embodiments the cell is a eukaryotic cell, such as a mammalian cell.

[0284] In some embodiments the cell in an immune effector cell.

[0285] In some embodiments the immune effector cell is selected from the group comprising: alphabeta T-cells, gamma-delta T-cells, tumour infiltrating lymphocytes (TILS), TCR-engineered T-cells, CAR T-cells, NK cells, NK / T cells, T regulatory cells, mucosal-associated invariant T-cells (MAIT), monocytes and macrophages.

[0286] In some embodiments the cell is a T-cell.

[0287] In some embodiments the cell is a CAR T-cell.

[0288] In some embodiments the cell is a stem cell or a progenitor cell.

[0289] In some embodiments the cell is a pluripotent stem cell, such as an embryonic and / or an induced pluripotent stem cell.

[0290] In some embodiments the cell is a multipotent stem cell, such as a haematopoietic stem cell

[0291] In some embodiments the cell may be derived from a subject, such as a human subject. In some embodiments the human subject may be a healthy human subject.

[0292] In some embodiments the human subject may be suffering from a disease. In this case the “subject” may be referred to as a “patient”.

[0293] Herein any method of transfection or transduction known in the art may be applied.

[0294] Composition

[0295] The invention includes a composition comprising the engineered donor cell, nucleic acid construct, antibody, nucleic acid construct, DNA molecule, plasmid or vector of the invention.

[0296] In some embodiments the composition may be a pharmaceutically acceptable composition.

[0297] In some embodiments the composition may comprise a pharmaceutically acceptable carrier, excipient, diluent or salt.

[0298] Methods of treatment

[0299] Any of the products of the invention, i.e. the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of the invention may be used to treat a disease.

[0300] Herein it will be understood that treatments falling within the scope of the invention encompass the in vivo delivery of products of the invention. In vitro delivery of products of the invention in order to produce engineered donor cells may also be considered a treatment.

[0301] The invention therefore contemplates the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of the invention for use in a method of treating a disease.

[0302] The invention also contemplates a method of treating a disease comprising administering the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of the invention to a subject in need thereof.

[0303] The invention also contemplates the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of the invention for use in the manufacture a medicament for the treatment of a disease. Within the invention the disease may be any disease which the skilled person understands may be treated by the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of the invention.

[0304] Herein the term “treatment” is intended to include therapeutic interventions that ameliorate a disease as well as curative treatments. Further, prophylactic use is also encompassed, such that said treatment may include treating a subject that is susceptible to a disease or is otherwise showing signs of progression towards a disease state without necessarily having symptoms of the disease or a clinical diagnosis of the disease.

[0305] Herein a subject to be treated according to the invention may be any subjection, including but not limited to a human subject such as a human adult subject, a human juvenile subject and a human infant subject.

[0306] In some embodiments the disease to be treated may be selected from the list consisting of cancer, an infectious disease, an auto-immune disease or an inherited disorder.

[0307] In some embodiments the cancer may be a T-cell malignancy, such as leukaemia or lymphoma, NK cell malignancy or CD7 positive acute myeloid leukaemia (AML).

[0308] Recently it has been observed that up to 30% of patients of myeloid cells express CD7. These patients also respond poorly to chemotherapy and standard of care (https: / / pubmed.ncbi.nlm.nih.gov / 30391141 / ).

[0309] In some embodiments the disease may be a CD7+ cell-related disease.

[0310] In some embodiments the CD7+ cell-related disease may be systemic sclerosis (https: / / pubmed.ncbi.nlm.nih.gov / 38123919 / ).

[0311] NUMBERED EMBODIMENTS OF THE INVENTION

[0312] Listed below are a set of numbered embodiments of the invention. Although these provide embodiments of the invention, they should not be considered limiting upon the scope of the invention.

[0313] 1. An engineered donor cell expressing a CD7-targeting polypeptide and one or more microRNA (miRNA) that functionally down-regulates a target in the cell. 2. The engineered donor cell of embodiment 1, wherein the CD7-targeting polypeptide specifically binds to CD7, optionally wherein the CD7-targeting polypeptide is an engineered T-cell receptor targeting CD7, optionally a chimeric antigen receptor (CAR) targeting CD7.

[0314] 3. The engineered donor cell of embodiment 1 or 2, wherein the miRNA improves the expansion of the cell, reduces the alloreactivity of the cell, reduces the immune system rejection of the cell, reduces the lymphodepletion of the cell, reduces the fratricide of the cell and / or improves the potency of the cell.

[0315] 4. The engineered donor cell of any of embodiments 1 to 3, wherein the miRNA functionally down-regulates TCR, CD7, HLA-I and / or HLA-II.

[0316] 5. The engineered donor cell of any of embodiments 1 to 4, wherein the miRNA functionally down-regulates CD7 by inhibiting the expression of CD7.

[0317] 6. The engineered donor cell of any of embodiments 1 to 5, wherein the miRNA functionally down-regulates TCR by inhibiting the expression of TCRa, TCRb, CD3d, CD3g, CD3e and / or CD3z.

[0318] 7. The engineered donor cell of any of embodiments 1 to 6, wherein the miRNA functionally down-regulates HLA-I by inhibiting the expression of one or more of a HLA-I polypeptide, B2M, NLRC5, TAPI, TAP2, TAPBP, RFX5, RFXANK, and / or RFXAP.

[0319] 8. The engineered donor cell of any of embodiments 1 to 7, wherein the miRNA functionally down-regulates HLA-II by inhibiting the expression of one or more of a HLA-II polypeptide, CIITA, RFX5, RFXANK, and / or RFXAP.

[0320] 9. The engineered donor cell of any of embodiments 1 to 8, wherein the miRNA functionally down-regulates CD5, B2M, CD3z, CD3e, CD3d, CD3g, TCRa, TCRb, CIITA, RFX5, RFXANK, RFXAP, TIM-3, PD-1, CD52, TIGIT, LAG-3, CTLA-4, DNMT3A, NR4A3, PRDM1, TGFBR2, REGNASE-1, ROQUIN-1, CCR5 and / or GM-CSF by inhibiting the expression thereof.

[0321] 10. The engineered donor cell of any of embodiments 1 to 9, wherein one or more surface- expressed polypeptide involved in immune signalling is up-regulated, optionally by a transcript expressing the polypeptide. 11. The engineered donor cell of embodiment 10, wherein the up-regulated surface- expressed polypeptide involved in immune signalling is a non-classical HLA class I polypeptide, CD47 and / or PD-L1.

[0322] 12. The engineered donor cell of any of embodiments 1 to 11, wherein a surface expressed non-classical HLA class I is up-regulated and a surface-expressed HLA class I is down- regulated.

[0323] 13. The engineered donor cell of embodiment 12, wherein the non-classical HLA class I polypeptide is a genetically modified HLA-E, HLA-G, or HLA-F polypeptide.

[0324] 14. The engineered donor cell of any of embodiments 1 to 13, wherein a surface expressed CAR is up-regulated and a surface expressed TCR is down-regulated.

[0325] 15. The engineered donor cell of any of embodiments 1 to 14, further expressing a safety switch gene or suicide gene.

[0326] 16. A nucleic acid construct comprising a sequence encoding a CD7-targeting polypeptide and one or more miRNA hairpins.

[0327] 17. The nucleic acid construct of embodiment 16, comprising a miRNA hairpin that:

[0328] (a) targets a sequence selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 1 to 128 or 282-293; and / or

[0329] (b) comprises a sequence selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 142 to 281.

[0330] 18. The nucleic acid construct of embodiment 16 or 17, wherein the CD7-targeting polypeptide comprises a polypeptide encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134, and / or wherein the CD7-targeting polypeptide comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140. 19. The nucleic acid construct of any of embodiments 16 to 18, wherein there are two copies of the first miRNA hairpin and / or two copies of the second miRNA hairpin.

[0331] 20. The nucleic acid construct of any of embodiments 16 to 19, wherein there are three copies of the first miRNA hairpin and / or three copies of the second miRNA hairpin.

[0332] 21. The nucleic acid construct of any of embodiments 16 to 20, wherein the construct comprises at least two different miRNA hairpins which target different regions of the same transcript.

[0333] 22. The nucleic acid construct of any of embodiments 16 to 21, wherein the construct comprises at least two different miRNA hairpins which target different transcripts of the same gene.

[0334] 23. The nucleic acid construct of any of embodiments 16 to 22, wherein the construct comprises at least two different miRNA hairpins which target different splice variants of the same gene.

[0335] 24. The nucleic acid construct of any of embodiments 16 to 23 further comprising a promoter element configured to express the miRNA hairpins, optionally wherein the promoter element is a promoter.

[0336] 25. The nucleic acid construct of embodiment 24, wherein the promoter is a eukaryotic promoter, optionally wherein the eukaryotic promoter is a Pol II or Pol III promoter.

[0337] 26. The nucleic acid construct of embodiment 24, wherein the promoter is an inducible promoter, a tissue-specific promoter, a cell lineage-specific promoter or a synthetic promoter.

[0338] 27. The nucleic acid construct of embodiment 24, wherein the promoter is a UBI promoter.

[0339] 28. The nucleic acid construct of embodiment 24, wherein the promoter is an EFla promoter, a derivative of an EFla promoter or an EFl short promoter, preferably wherein the promoter is an EF 1 short promoter.

[0340] 29. The nucleic acid construct of any of embodiments 16 to 28, further comprising a promoter element configured to express the CD7-targeting polypeptide, optionally wherein the promoter element is a promoter. 30. The nucleic acid construct of embodiment 29, wherein the promoter configured to express the CD7-targeting polypeptide is a PGK promoter, an EFla promoter, a derivative of an EFla promoter or an EFl short promoter.

[0341] 31. The nucleic acid construct of any of embodiments 16 to 30, further comprising a spacer, optionally wherein the spacer comprises an enhancer, further optionally wherein the spacer is an enhancer.

[0342] 32. The nucleic acid construct of embodiment 31, wherein the spacer is at least 50 nucleotides in length.

[0343] 33. The nucleic acid construct of embodiment 31, wherein the spacer is between 50 and 1,000 nucleotides in length.

[0344] 34. The nucleic acid construct of embodiment 31, wherein the spacer is between 50 and 900, 50 and 800, 100 and 800, or 50 and 800 nucleotides in length.

[0345] 35. The nucleic acid construct of embodiment 31, wherein the spacer is at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 200 nucleotides in length.

[0346] 36. The nucleic acid construct of any of embodiments 31 to 35, wherein the spacer is a GFP sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 141.

[0347] 37. The nucleic acid construct of any of embodiments 31 to 36, wherein the spacer is positioned between the promoter configured to express the miRNA hairpins and the miRNA hairpins.

[0348] 38. The nucleic acid construct of any of embodiments 31 to 37, wherein the spacer is heterologous with respect to the promoter element.

[0349] 39. The nucleic acid construct of any of embodiments 31 to 38, wherein the spacer comprises an encoded open reading frame.

[0350] 40. The nucleic acid construct of any of embodiments 16 to 39, wherein at least two of the miRNA hairpins are separated by an intervening sequence. 41. The nucleic acid construct of any of embodiments 16 to 40, wherein the CD7-targeting polypeptide specifically binds to CD7, optionally wherein the CD7-targeting polypeptide is an engineered T-cell receptor targeting CD7, optionally a CAR targeting CD7.

[0351] 42. The nucleic acid construct of any of embodiments 16 to embodiment 41, wherein the sequence encoding a CD7-targeting polypeptide comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134, and / or wherein the CD7-targeting polypeptide comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

[0352] 43. The nucleic acid construct of any of embodiments 16 to 42, wherein the miRNA hairpins are under the control of a first promoter and the sequence encoding a CD7-targeting polypeptide is under the control of a second promoter, or wherein the miRNA hairpins and the sequence encoding a CD7-targeting polypeptide are under the control of a single promoter.

[0353] 44. The nucleic acid construct of any of embodiments 16 to 43, wherein the construct further comprises a selection gene.

[0354] 45. The nucleic acid construct of embodiment 45, wherein the selection gene is LNGFR, truncated endothelial growth factor receptor (tEGFR), tCD19, CD20 or a truncated CD20 (tCD20), tCD34 or a derivative thereof.

[0355] 46. The nucleic acid construct of any of embodiments 16 to 45, wherein the construct further comprises a sequence encoding a suicide gene or safety switch gene.

[0356] 47. The nucleic acid construct of embodiment 46, wherein the suicide gene or safety switch gene is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), inducible caspase 9 (iCasp9), truncated endothelial growth factor receptor (tEGFR), RQR8, dihydrofolate reductase (DHFR), CD20 or a truncated CD20 (tCD20), thymidylate synthase (TYMS) and lenalidomide OFF-switch CAR molecules.

[0357] 48. The nucleic acid construct of any of embodiments 16 to 47, wherein the construct further comprises an internal ribosome entry site (IRES). 49. A DNA molecule, plasmid or vector comprising the nucleic acid construct of any of embodiments 16 to 48, optionally wherein the vector is an expression vector, optionally wherein the expression vector is an adenovirus, an adeno-associated virus, a retrovirus or lentivirus vector.

[0358] 50. A method for preparing an engineered donor cell comprising transfecting or transducing a cell with the nucleic acid construct, DNA molecule, plasmid or vector of any of embodiments 16 to 49.

[0359] 51. A method for preparing an engineered donor cell from a patient donor or healthy donor comprising:

[0360] (a) collecting a cell from the donor;

[0361] (b) transfecting or transducing the cell with the nucleic acid construct, DNA molecule, plasmid or vector of any of embodiments 16 to 49; and

[0362] (c) expressing the nucleic acid construct.

[0363] 52. The method of embodiment 50 or 51, wherein the engineered donor cell is a T-cell.

[0364] 53. The method of any of embodiments 50 to 52, wherein the nucleic acid construct, DNA molecule, plasmid or vector down-regulates a TCR polypeptide and up-regulates a CAR polypeptide targeting CD7, wherein the engineered donor cell is a CAR T-cell.

[0365] 54. An engineered donor cell obtainable or obtained by the method of any of embodiments 50 to 53.

[0366] 55. A cell comprising the nucleic acid construct, DNA molecule, plasmid or vector of any of embodiments 1 to 49, optionally wherein the cell is an engineered donor cell.

[0367] 56. The engineered donor cell of any of embodiments 1 to 15, 54 or 55, wherein the engineered donor cell is a eukaryotic cell, optionally wherein the engineered donor cell is a mammalian cell.

[0368] 57. The engineered donor cell of any of embodiments 1 to 15 or 54 to 56, wherein the engineered donor cell in an immune effector cell. 58. The engineered donor cell of embodiment 57, wherein the immune effector cell is selected from the group comprising: alpha-beta T-cells, gamma-delta T-cells, tumour infiltrating lymphocytes (TILS), TCR-engineered T-cells, CAR T-cells, NK cells, NK / T-cells, T regulatory cells, mucosal-associated invariant T-cells (MAIT), monocytes and macrophages.

[0369] 59. The engineered donor cell of embodiment 58, wherein the immune effector cell is a CAR T-cell.

[0370] 60. The engineered donor cell of any of embodiments 1 to 15 or 54 to 56, wherein the engineered donor cell is a stem cell or a progenitor cell.

[0371] 61. The engineered donor cell of embodiment 60, wherein the engineered donor cell is a pluripotent stem cell, such as an embryonic and / or an induced pluripotent stem cell.

[0372] 62. The engineered donor cell of embodiment 60, wherein the engineered donor cell is a multipotent stem cell, such as a haematopoietic stem cell.

[0373] 63. The engineered donor cell of any of embodiments 1 to 15 or 54 to 56, wherein the engineered donor cell is a differentiated cell.

[0374] 64. A composition comprising the engineered donor cell of any of embodiments 1 to 15 or 54 to 63, optionally further comprising a pharmaceutically acceptable carrier, excipient, diluent or salt.

[0375] 65. An in vitro, ex vivo or in vivo method of depleting CD7+ cells, comprising contacting the CD7+ cells with the engineered donor cell or composition of any of embodiments 1 to 15 or 54 to 64.

[0376] 66. The engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of any of embodiments 1 to 49 or 54 to 64, for use in a method of treating a disease.

[0377] 67. A method of treating a disease comprising administering the engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of any of embodiments 1 to 49 or 54 to 64 to a subject in need thereof.

[0378] 68. The engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of any of embodiments 1 to 49 or 54 to 64, for use in the manufacture a medicament for the treatment of a disease. 69. The method of any of embodiments 66 to 68, wherein the disease is selected from the list consisting of cancer, an infectious disease, an auto-immune disease or an inherited disorder.

[0379] 70. The method of embodiment 69, wherein the cancer is a T-cell malignancy, such as leukaemia or lymphoma.

[0380] 71. The method of any of embodiments 66 to 70, wherein the disease is a CD7+ cell-related disease.

[0381] 72. A miRNA hairpin, wherein the miRNA hairpin targets a sequence selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 1 to 128 or 282-293.

[0382] 73. A miRNA hairpin, wherein the miRNA hairpin comprises a sequence selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 142 to 281.

[0383] 74. A VHH antibody or antibody fragment, wherein the VHH antibody or antibody fragment targets a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 135 to 140.

[0384] 75. An engineered donor cell or nucleic acid construct as substantially described herein with reference to and as illustrated by the accompanying drawings.

[0385] EXAMPLES

[0386] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1

[0387] Gene silencing constructs require (i) target sequence design, (ii) molecular cloning to create single hairpin miRNA constructs and lentiviral vector production, and (iii) gene modification of target cells and assessment of gene / protein silencing using appropriate read-out methods. Optimal target sequence(s) may then be used to create multi-hairpin miRNA constructs to similarly assess for maximal gene silencing efficiencies. Once optimal gene silencing constructs have been developed, they are incorporated within CAR-expressing constructs for further validation and functional testing in CAR T-cell products.

[0388] For the development of novel CAR constructs, single-domain, heavy-chain only (VHH) binders were used as the targeting moiety of the CAR. Single-domain binders offer notable benefits for CAR engineering when compared to the traditionally used single chain variable fragments (scFv), which include: (i) improved stability, given that there is no need for the assembly of two different variable chains; and (ii) smaller size by up to 40% (15 vs 25 kDa), allowing for more compact payloads, improved vector production and more efficient engineering of target cells. In particular, fully humanized VHH binders (Moutel et al., 2016) were used for the CAR designs, which decrease the likelihood of CAR immunogenicity, and therefore the risk of host rejection in initial and subsequent re-dosing of allogeneic CAR T- cells.

[0389] Target sequence design

[0390] Target sequences for incorporation into miRNA architecture were designed using an in-house developed pipeline that prioritizes the selection of optimal gene silencing target sequences based on predefined set of calculated features. The backbone of the pipeline was developed in python with reference sequences obtained from the current Ensembl version. The potential / likelihood of gene silencing of each potential target sequence is predicted by assessing it against a number of molecular features, including but not limited to, the presence of specific nucleotide compositions, transcript availability, and the number of inverted repeats. Sequence prioritization was based on identifying conserved regions in target transcripts, individually ranking each target sequence and mitigating the risk of potential off-target gene silencing (based on sequence identity between the target sequence and the transcriptome of the target cells). The notation “TARGET NAME T#” is used to refer to target sequences on the mRNA of the genes, and the associated miRNA targeting those sequences. VHH binder discovery

[0391] Anti-hCD7 VHH were discovered using a synthetic antibody library with a complexity of 3 billion VHHs. Three rounds of Phage display selection were performed using biotinylated hCD7, which allowed selection of VHHs that recognise the non-adsorbed antigen in a native form. Selected VHHs were then validated in non-adsorbed Phage ELISA and non-adsorbed induced ELISA.

[0392] Molecular cloning

[0393] Prioritized target sequences were synthesized within a mirGE backbone by a third-party manufacturer. The single mirGE sequences were cloned using LR Clonase II. The mirGE pENTR plasmid, an elongation factor 1 short promoter (pENTR-L4- EFs-LlR) plasmid and a lentivector destination cassette (pCWX-R4dESTR2-PC) containing the mCherry reporter gene or CAR molecule were cloned into a single plasmid. Successful cloning of all constructs was confirmed via restriction enzyme digestion pattern and DNA sequencing.

[0394] Lentiviral vectors and titration

[0395] Lentiviral vectors carrying the mCherry reporter gene were produced by transfecting HEK293T cells with transfer plasmids carrying the gene silencing construct, as well as lentiviral packaging (PAX2) and envelope (VSVg) plasmids. The cell culture medium was replenished after 4-6 hours and subsequently harvested at 24 hours for viral particle collection. Lentiviral vectors for CAR constructs were produced by transfecting Viral Production Cells (ThermoFisher), a derivative of the HEK293F cell line, with transfer plasmids carrying the gene silencing and CAR construct, as well as lentiviral packaging (PAX2) and envelope (VSVg) plasmids. The cell culture medium was harvested at 48 hours post-transfection for viral particle collection. For all lentiviral vectors, the culture medium was collected, filtered to remove cellular debris, and viral particles enriched using PEG-It Virus Precipitation Solution (System Biosciences), according to the manufacturer’s instructions. Final aliquots of concentrated lentiviral vectors were stored at -80°C. Functional viral vector titres were assessed by transducing HT1080 cells over a range of dilutions and measuring the percentage of cells expressing mCherry or RQR8 reporter gene.

[0396] Cells and cell lines The expression and silencing of all gene constructs was assessed in primary T-cells, which were prepared from anonymized huffy coat blood units procured from the Blood Transfusion Centre of the University Hospital of Geneva, Switzerland. The peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll separation, after which T-cells were separated using the EasySep™ Human T-cell Isolation Kit (StemCell) and cryopreserved in aliquots in liquid nitrogen.

[0397] Gene delivery and CAR T-cell manufacturing

[0398] Cryopreserved T-cells were thawed, cultured in T-cell medium (X vivo, 5% Human Serum, 1% penicillin / streptomycin) complemented with IL-7 and IL-15 (lOng / mL each), and activated using CD3 / CD28 Dynabeads, as per manufacturer’s instructions. Activated T-cells were transduced 24-48 hours later with lentiviral vectors carrying the miRNA gene silencing constructs (for mirGE screening) and miRNA gene silencing and CAR expression constructs (for CAR T-cell production). Transductions were performed in high density volumes (2 million cells per mL in regular culture plates for mirGE screening and 1 million cells per mL for CAR T-cell production in GREX plates). For mirGE screening, the medium was exchanged for fresh medium after 24h and every other day thereafter for T-cell maintenance at a cell density 1 million per mL, for a minimum of 5 days prior to assessing silencing of target genes. For CAR T-cell production, medium was added to top up the well 24h after transduction and 8 days after transduction cells were re-seeded in a new GREX plate (in a 24-well GREX to re-seed between 0.5-1 million cells and in a 6-well GREX to re-seed between 5-15 million cells). When cells were re-seeded in a 24-well GREX, half of the medium was exchanged on D12 for fresh medium. For CAR T-cell production and on day 15 after transduction, TCR depletion (EasySep™ Human TCR Alpha / Beta Depletion Kit, StemCell) was performed and cells were frozen and stored in liquid nitrogen. During this production process, silencing, transduction and memory, exhaustion and activation markers were assessed by flow cytometry at day 5 and 17 after transduction and post-thaw.

[0399] Flow cytometry and assessment of gene silencing

[0400] Flow cytometry was performed at 5 days post-transduction for cells produced for mirGE screening and at 5 and 17 days post-transduction, and post-thaw for CAR T-cells. Cells were harvested, washed, re-suspended in Staining buffer (EasySep™ Human TCR Alpha / Beta Depletion Kit (Miltenyi Biotec), 5% BSA), and stained for 10-30 min with the appropriate antibodies for assessment of cell surface expression. Following staining, cells were washed with Staining buffer, re-suspended in Staining buffer, and cell surface expression assessed via flow cytometry. Captured data were exported to FlowJo for analysis. To calculate the level of gene silencing, changes in both the percentage of cells positive for the target and median florescence intensity (MFI) were assessed. Normalization includes expression levels within samples (modified vs unmodified cells), after which expression levels relative to the control- transduced cells were calculated.

[0401] Cytotoxicity assays

[0402] Cell lines MOLT-4 (ATCC CRL-1582) and SUP-T1 (ATCC CRL-192) were purchased from ATCC. Tumor cells stained with Cell Trace Violet (Thermo Fisher Scientific) or stably expressing GFP were mixed with un-transduced or CAR T-cells at effector to target ratios 1 : 1, 1 :3 or 1 :9. For short-term killing assays, the number of tumor and T-cells were assessed by flow cytometry after 24h, 48h and 72h. For recursive killing assays, the number of tumor cells and T-cells was assessed every 3-4 days by flow cytometry. At each time point and upon tumor cell clearance, the same number as initially plated of new tumor cells was added to T-cells.

[0403] In vivo model of tolerability

[0404] Seven to nine-week-old non-obese diabetic (NOD)-Cg-PrkdcscidI12rgtml Wjl / SzJ (NSG) mice were purchase from Charles River UK. All procedures were done in compliance with the UK Animal Scientific Procedures Act 1986 (ASP A). CAR T cells were prepared as described previously. Each mouse (weighting >15g at study initiation) received a single intravenous injection of 7.5 x 106CAR T cells in a total volume of 100 pL via the tail vein. Mice were monitored daily for general health, behaviour, and signs of adverse effects. Body weight was measured daily and recorded as an indicator of tolerability. Relative body weight was calculated as a percentage of the initial body weight on Day 0 (baseline). The animals were followed for a total of 15 days post-injection.

[0405] In vivo model of tumor cell engraftment

[0406] Seven to nine-week-old non-obese diabetic (NOD)-Cg-PrkdcscidI12rgtmlWjl / SzJ (NSG) mice were purchase from Charles River UK. All procedure were done in compliance with the UK Animal Scientific Procedures Act 1986 (ASP A). Mice were intravenously injected with 0.3 xlO6CCRF-CEM / GFP-ffluc, followed by a single injection of 0.2 x 106, 1.5 x 106or 6.5 x 106 miCAR7 or 6.4 x 106miCAR19 T-cells 5 days later. From day 4 onwards, tumor burden was monitored using an IVIS Spectrum CT Imaging system (Perkin Elmer) by recording bioluminescence from mice intraperitoneally injected with 150 mg per kg of d-Luciferin, twice weekly. Living Image software (Caliper LS, US) was used to visualize and quantify total luminescence.

Claims

CLAIMS1. An engineered donor cell expressing a CD7-targeting polypeptide and one or more microRNA (miRNA) that functionally down-regulates a target in the cell.

2. The engineered donor cell of claim 1, wherein the CD7-targeting polypeptide specifically binds to CD7, optionally wherein the CD7-targeting polypeptide is an engineered T-cell receptor targeting CD7, optionally a chimeric antigen receptor (CAR) targeting CD7.

3. The engineered donor cell of claim 1 or 2, wherein the miRNA functionally down- regulates TCR, CD7, HLA-I and / or HLA-II.

4. The engineered donor cell of any of claims 1 to 3, wherein the miRNA functionally down-regulates CD7 by inhibiting the expression of CD7.

5. The engineered donor cell of any of claims 1 to 4, wherein the miRNA functionally down-regulates CD5, B2M, CD3z, CD3e, CD3d, CD3g, TCRa, TCRb, CIITA, RFX5, RFXANK, RFXAP, TIM-3, PD-1, CD52, TIGIT, LAG-3, CTLA-4, DNMT3A, NR4A3, PRDM1, TGFBR2, REGNASE-1, ROQUIN-1, CCR5 and / or GM-CSF by inhibiting the expression thereof.

6. The engineered donor cell of any of claims 1 to 5, further expressing a safety switch gene or suicide gene.

7. A nucleic acid construct comprising a sequence encoding a CD7-targeting polypeptide and one or more miRNA hairpins.

8. The nucleic acid construct of claim 8, comprising a miRNA hairpin that:(a) targets a sequence selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 1 to 128 or 282-293; and / or(b) comprises a sequence selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 142 to 281.

9. The nucleic acid construct of claim 7 or 8, wherein the CD7-targeting polypeptide comprises a polypeptide encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129, SEQ ID NO: 130,SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134, and / or wherein the CD7-targeting polypeptide comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

10. The nucleic acid construct of any of claims 7 to 9 further comprising a promoter element configured to express the miRNA hairpins, optionally wherein the promoter element is a promoter and / or a promoter element configured to express the CD7-targeting polypeptide, optionally wherein the promoter element is a promoter.

11. The nucleic acid construct of any of claims 7 to 10, further comprising a spacer, optionally wherein the spacer comprises an enhancer, further optionally wherein the spacer is an enhancer.

12. The nucleic acid construct of claim 11, wherein the spacer is a GFP sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 141.

13. The nucleic acid construct of any of claims 7 to 12, wherein the CD7-targeting polypeptide specifically binds to CD7, optionally wherein the CD7-targeting polypeptide is an engineered T-cell receptor targeting CD7, optionally a CAR targeting CD7.

14. The nucleic acid construct of any of claims 7 to 13, wherein the sequence encoding a CD7-targeting polypeptide comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134, and / or wherein the CD7-targeting polypeptide comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

15. The nucleic acid construct of any of claims 7 to 14, wherein the construct further comprises a sequence encoding a suicide gene or safety switch gene.

16. The nucleic acid construct of claim 15, wherein the suicide gene or safety switch gene is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), inducible caspase 9 (iCasp9), truncated endothelial growth factor receptor (tEGFR), RQR8,dihydrofolate reductase (DHFR), CD20 or a truncated CD20 (tCD20), thymidylate synthase (TYMS) and lenalidomide OFF-switch CAR molecules.

17. A DNA molecule, plasmid or vector comprising the nucleic acid construct of any of claims 7 to 16, optionally wherein the vector is an expression vector, optionally wherein the expression vector is an adenovirus, an adeno-associated virus, a retrovirus or lentivirus vector.

18. A method for preparing an engineered donor cell comprising transfecting or transducing a cell with the nucleic acid construct, DNA molecule, plasmid or vector of any of claims 7 to 17.

19. A method for preparing an engineered donor cell from a patient donor or healthy donor comprising:(a) collecting a cell from the donor; and(b) transfecting or transducing the cell with the nucleic acid construct, DNA molecule, plasmid or vector of any of claims 7 to 18; and(c) expressing the nucleic acid construct.

20. An engineered donor cell obtainable or obtained by the method of claim 18 or claim 1921. The engineered donor cell of claim 20, wherein the immune effector cell is selected from the group comprising: alpha-beta T-cells, gamma-delta T-cells, tumour infiltrating lymphocytes (TILS), TCR-engineered T-cells, CAR T-cells, NK cells, NK / T-cells, T regulatory cells, mucosal-associated invariant T-cells (MAIT), monocytes and macrophages.

22. The engineered donor cell, nucleic acid construct, DNA molecule, plasmid, vector or composition of any of claims 1 to 21, for use in a method of treating a disease.

23. The method of any claim 22, wherein the disease is selected from the list consisting of cancer, an infectious disease, an auto-immune disease or an inherited disorder.

24. A miRNA hairpin, wherein the miRNA hairpin:(a) target a sequence selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 1 to 128 or 282-293; and / or(b) comprises a sequence selected from sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOs: 142 to 281.

25. A VHH antibody or antibody fragment, wherein the VHH antibody or antibody fragment targets a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 1 to 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134, and / or wherein the VHH antibody or antibody fragment targets a polypeptide encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139 or SEQ ID NO: 140.

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