Lentiviral vectors

Abstract

The present invention relates to a lentiviral vector that encodes a methylmalonic acid (MMA) associated polypeptide, and a method for preparing the lentiviral vector. The invention also relates to a cell and a pharmaceutical composition comprising said lentiviral vector, and to the use of said lentiviral vector in the treatment of methylmalonatemia (MMA).

Description

Technical Field

[0001] The present invention relates to a lentiviral vector encoding a methylmalonic acidemia (MMA)-associated polypeptide, a cell and a pharmaceutical composition comprising the lentiviral vector, and the use of the lentiviral vector in treating methylmalonic acidemia (MMA). Background Art

[0002] Methylmalonic acidemia (MMA), also known as methylmalonic aciduria, is a group of inborn errors of metabolism associated with elevated concentrations of methylmalonic acid in the blood and urine, which may result from the failure to convert methylmalonyl-CoA to succinyl-CoA during propionyl-CoA metabolism in the mitochondrial matrix.

[0003] Isolated MMA (iMMA) can refer to MMA without changes in other metabolites and can be caused by: complete (mut) 0 Enzymatic isoform) or part (mut – The disorder can be caused by a defect in the transport or synthesis of its cofactor adenosylcobalamin (caused by pathogenic variants in MMAA, MMAB, or MMADHC); or a defect in the enzyme methylmalonyl-CoA epimerase (encoded by MCEE) (see, e.g., Manoli I, et al. 2005. Isolated Methylmalonic Acidemia. In Gene Reviews).

[0004] The onset of MMA manifestations ranges from the neonatal period to adulthood. Affected children often display anorexia, failure to thrive, hypotonia, developmental delay, progressive renal failure, functional immune impairment, optic atrophy, and hematologic abnormalities. For example, patients with the most severe form of iMMA (mut 0 ) patients, show symptoms such as lethargy, vomiting, respiratory distress, severe ketoacidosis and hyperammonemia in the neonatal period. Patients with MMA experience significant morbidity and mortality, and the prognosis for long-term survival is poor (see, for example, Zhou, X., et al., 2018. Intractable & rare disorders research, 7 (2), pp. 73-78).

[0005] The treatment of MMA usually consists of a protein-restricted diet and drug management of symptoms. However, patients are still at risk of recurrent episodes of life-threatening metabolic crises and multi-organ diseases, disability and eventual death (see, for example, Fraser, JL and Venditti, CP, 2016. Current opinion in pediatrics, 28 (6), pp. 682-693). In particular, patients with iMMA usually have earlier and more severe clinical manifestations, most of which are accompanied by long-term complications and recurrent metabolic decompensation involving multiple organs. Liver transplantation is currently the only effective treatment for MMA patients with recurrent metabolic decompensation (see, for example, Jiang, YZ and Sun, LY, 2019. Frontiers in Pediatrics, 7, p. 87).

[0006] Therefore, there is a need for new methods of treating or preventing MMA, such as iMMA.

[0007] Lentiviral vectors encoding methylmalonyl-CoA mutase are described in Wong, ES, et al., 2014. Human gene therapy, 25(6), pp. 529-538. To test the lentiviral vectors, Wong et al. used adult Mut – / – MUT h2 Mice that have a mouse MUT knockout but express two copies of human MUT, thereby displaying a mild phenotype. Since these mice are transgenic for human MUT, they are immune tolerant to human MUT and therefore cannot properly assess possible immune responses triggered by lentiviral gene transfer. Such immune responses are a long-established problem for lentiviral gene therapy (see, e.g., VandenDriessche, T., et al., 2007. Journal of Thrombosis and Haemostasis, 5(1), pp. 16-24). Summary of the invention

[0008] The inventors have developed gene therapies for the treatment and / or prevention of MMA, such as iMMA. The lentiviral vector-mediated gene therapy described herein can allow stable gene transfer in pediatric patients even in the first disease stage by virtue of lentiviral vector genome integration, and thus can alleviate symptoms and limit progressive damage to hepatocytes.

[0009] The inventors have surprisingly shown that the lentiviral vector-mediated gene therapy described herein is safe and effective in the MMA model. The inventors have generated a lentiviral vector encoding a human codon-optimized MMUT coding sequence under the control of a hepatocyte-specific cassette. Administration of the lentiviral vector resulted in a significant decrease in serum methylmalonic acid, indicating improved amino acid metabolism.

[0010] Immunoshielded lentiviral vectors can reduce immune responses after administration, so that there is no loss of therapeutic effect over time. Targeting transgene expression to hepatocytes (e.g., using one or more miRNA target sequences and / or hepatocyte-specific promoters) can reduce immune responses in the context of MUT knockout mice in the absence of any expression of human transgenes. In contrast, as described above, Wong et al. administered lentiviral vectors to mice that were transgenic for human MUT and were therefore immune tolerant to expression of the human transgene.

[0011] In addition, the inventors have demonstrated that the lentiviral vector of the present invention is effective in juveniles. The inventors have shown that after administration to young mice (2 weeks old) with hepatocyte proliferation, stable therapeutic effects (e.g., significant reduction in circulating MMA) last up to 12 months after gene therapy. Even with integrative vectors, it is not always desirable to obtain stable therapeutic effects in juveniles. If the integrative vector does not transduce the hepatocytes responsible for generating adult livers after growth and / or if there is a counter-selection of corrected hepatocytes, a stable therapeutic effect may not be obtained. In contrast, Wong et al. treated adult mice.

[0012] Thus, in contrast to Wong et al., the present inventors have demonstrated that the lentiviral vectors of the present invention are effective in immatures in a model that exhibits a more severe phenotype and is not immunologically tolerant to expression of a human transgene.

[0013] In one aspect, the present invention provides a lentiviral vector comprising a nucleotide sequence encoding a methylmalonic acidemia (MMA)-associated polypeptide. The lentiviral vector may be an immune-shielded lentiviral vector.

[0014] The MMA-associated polypeptide can be selected from methylmalonyl-CoA mutase (MMUT) or a fragment thereof; methylmalonic aciduria type A (MMAA) or a fragment thereof; methylmalonic aciduria type B (MMAB) or a fragment thereof; methylmalonic aciduria and homocystinuria type D (MMADHC) or a fragment thereof; and methylmalonyl-CoA epimerase (MCEE) or a fragment thereof. In a preferred embodiment, the MMA-associated polypeptide is MMUT or a fragment thereof.

[0015] Suitably, the MMA-associated polypeptide comprises or consists of an amino acid sequence at least 70% identical to one of SEQ ID NO: 37, 40, 42, 44 or 46, or a fragment thereof. In some embodiments, the MMA-associated polypeptide comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 37, or a fragment thereof.

[0016] Suitably, the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of a nucleotide sequence at least 70% identical to one of SEQ ID NOs: 38, 39, 41, 43, 45 or 47, or a fragment thereof. In some embodiments, the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of a nucleotide sequence at least 70% identical to one of SEQ ID NOs: 38 or 39, or a fragment thereof. Suitably, the nucleotide sequence encoding the MMA-associated polypeptide is codon-optimized. In some embodiments, the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of a nucleotide sequence at least 70% identical to SEQ ID NO: 39, or a fragment thereof.

[0017] Lentiviral vectors can be CD47 高 Lentiviral vector. Suitably, the lentiviral vector is obtained from CD47 高 A host cell, preferably wherein the host cell is genetically engineered to increase expression of CD47 on the cell surface. Suitably, the lentiviral vector has at least about 2-fold more CD47 on its surface than a lentiviral vector obtained from an unmodified host cell.

[0018] Lentiviral vectors can be MHC-I 无 Lentiviral vector. Suitably, the lentiviral vector is obtained from MHC-I 无 A host cell, preferably wherein the host cell is genetically engineered to disrupt expression of MHC-I on the cell surface. Suitably, the lentiviral vector has less than about 50% of the number of surface exposed MHC-I molecules displayed on the lentiviral vector obtained from an unmodified host cell. Suitably, MHC-I is not detectable on the surface of the lentiviral vector.

[0019] Lentiviral vectors can be CD47 高 / MHC-I 无 Lentiviral vector. Suitably, the lentiviral vector is obtained from CD47 高 / MHC-I 无Suitably, the lentiviral vector has at least about 2-fold more CD47 on its surface than a lentiviral vector obtained from an unmodified host cell and MHC-I is not detectable on the surface of the lentiviral vector.

[0020] The nucleotide sequence encoding the MMA-associated polypeptide can be operably linked to one or more miRNA target sequences. In some embodiments, the one or more miRNA target sequences inhibit transgene expression in one or more cells other than hepatocytes. In some embodiments, the one or more miRNA target sequences inhibit transgene expression in hematopoietic lineage cells and / or antigen presenting cells.

[0021] In some embodiments, one or more miRNA target sequences are selected from miR-181, miR-142, miR-223 and miR-155 target sequences. In some embodiments, the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to one or more mir-142 target sequences, two or more mir-142 target sequences, three or more mir-142 target sequences or four or more mir-142 target sequences. In some embodiments, the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to four mir-142 target sequences. In some embodiments, one or more miRNA target sequences include or consist of a nucleotide sequence at least 90% identical to SEQ ID NO: 17. In some embodiments, one or more miRNA target sequences include or consist of a nucleotide sequence at least 90% identical to SEQ ID NO: 18.

[0022] The nucleotide sequence encoding the MMA associated polypeptide can be operably connected to a liver-specific promoter. In some embodiments, the nucleotide sequence encoding the MMA associated polypeptide is operably connected to a hepatocyte-specific promoter. Suitably, the nucleotide sequence encoding the MMA associated polypeptide is operably connected to a transthyretin (TTR) promoter, an alpha-1-antitrypsin (AAT) promoter, a thyroxine binding globulin (TBG) promoter, an APoE / hAAT promoter, an HCR-hAAT promoter, an LP1 promoter, or an HLP promoter. In some embodiments, the nucleotide sequence encoding the MMA associated polypeptide is operably connected to a transthyretin (TTR) promoter. In a preferred embodiment, the nucleotide sequence encoding the MMA associated polypeptide is operably connected to an Enh1mTTR (ET) promoter. In some embodiments, the nucleotide sequence encoding the MMA associated polypeptide is operably connected to a promoter, and the promoter comprises or consists of a nucleotide sequence at least 70% identical to SEQ ID NO: 19.

[0023] The lentiviral vector may be pseudotyped. Suitably, the lentiviral vector is VSV.G pseudotyped. The lentiviral vector may be a self-inactivating (SIN) lentiviral vector. Suitably, the lentiviral vector comprises a self-inactivating (SIN) LTR comprising or consisting of a nucleotide sequence at least 70% identical to SEQ ID NO: 23 or a fragment thereof. The lentiviral vector may be an integrative lentiviral vector and / or a replication-defective lentiviral vector. The lentiviral vector may be HIV-derived.

[0024] In some embodiments, the lentiviral vector comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO:36.

[0025] In one aspect, the invention provides a method of producing a lentiviral vector according to the invention.

[0026] In one aspect, the present invention provides a kit or system for producing a lentiviral vector of the present invention.

[0027] In one aspect, the invention provides a cell comprising a lentiviral vector according to the invention. The cell may be an isolated cell.

[0028] In one aspect, the present invention provides a pharmaceutical composition comprising a lentiviral vector or a cell according to the present invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0029] In one aspect, the invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the invention for use as a medicament.

[0030] In one aspect, the present invention provides use of the lentiviral vector, cell or pharmaceutical composition according to the present invention in the preparation of a medicament.

[0031] In one aspect, the present invention provides a method comprising administering a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0032] In one aspect, the present invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the present invention for use in preventing or treating methylmalonic acidemia (MMA), such as isolated MMA (iMMA).

[0033] In one aspect, the present invention provides use of a lentiviral vector, a cell or a pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating methylmalonic acidemia (MMA), such as isolated MMA (iMMA).

[0034] In one aspect, the present invention provides a method for preventing or treating methylmalonic acidemia (MMA), such as isolated MMA (iMMA), comprising administering to a subject in need thereof a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention.

[0035] Suitably, the MMA-associated polypeptide is MMUT or a fragment thereof, and MMA is mut-type MMA (mut-MMA); wherein the MMA-associated polypeptide is MMAA or a fragment thereof, and MMA is cblA-type MMA (cblA-MMA); the MMA-associated polypeptide is MMAB or a fragment thereof, and MMA is cblB-type MMA (cblB-MMA); the MMA-associated polypeptide is MMADHC or a fragment thereof, and MMA is cblD-type MMA (cblD-MMA); or the MMA-associated polypeptide is MCEE or a fragment thereof, and MMA is due to MCEE deficiency. In a preferred embodiment, the MMA-associated polypeptide is MMUT or a fragment thereof, and MMA is mut-MMA.

[0036] The lentiviral vector, cell or pharmaceutical composition can be administered to any subject in need thereof. In some embodiments, the subject is a human subject. In some embodiments, the subject is a juvenile. In some embodiments, the subject is a pediatric patient. In some embodiments, the subject is a neonatal patient or an infant patient.

[0037] The lentiviral vector, cell or pharmaceutical composition can be administered by any suitable route. In some embodiments, the lentiviral vector, cell or pharmaceutical composition is administered systemically (e.g., by intravenous injection or intraperitoneal injection). In some embodiments, the lentiviral vector, cell or pharmaceutical composition is administered locally (e.g., by direct injection, intra-arterial injection or intra-portal vein injection). In some embodiments, the lentiviral vector, cell or pharmaceutical composition is administered locally to the liver (e.g., by intrahepatic injection, intra-hepatic artery injection or intra-portal vein injection).

[0038] The lentiviral vector, cell, or pharmaceutical composition can be administered at any suitable dose. In some embodiments, the lentiviral vector is administered at a dose of at least about 10 8 TU / kg, at least about 10 9 TU / kg or at least about 10 10 TU / kg. In some embodiments, the lentiviral vector is administered at a dose of about 10 8 to about 10 11 TU / kg, about 10 8 to about 10 10 TU / kg or about 10 9 to about 10 10 The dose of TU / kg was administered.

[0039] The lentiviral vector may be integrated into the genome of a liver cell and maintained as a liver cell replica. Suitably, the lentiviral vector is integrated into the genome of a hepatocyte and maintained as a hepatocyte replica.

[0040] Following administration, the subject's plasma methylmalonic acid levels and / or urine methylmalonic acid levels may be reduced and / or normalized; and / or the subject's mitochondrial function may be improved.

[0041] In one aspect, the invention provides an immunoshielded lentiviral vector for use in a method of therapy. In one aspect, the invention provides a cell for use in a method of therapy, wherein the cell comprises an immunoshielded lentiviral vector.

[0042] In one aspect, the present invention provides the use of an immune-shielded lentiviral vector in the preparation of a medicament for treating or preventing a disease. In one aspect, the present invention provides the use of a cell in the preparation of a medicament for treating or preventing a disease, wherein the cell comprises an immune-shielded lentiviral vector.

[0043] In one aspect, the invention provides a method of treatment, wherein the method comprises administering to a subject an immune-shielded lentiviral vector. In one aspect, the invention provides a method of treatment, wherein the method comprises administering to a subject a cell, wherein the cell comprises an immune-shielded lentiviral vector.

[0044] In one aspect, the invention provides an immune-shielded lentiviral vector for use in a method of therapy, wherein the method comprises administering the immune-shielded lentiviral vector to a minor or pediatric subject. In one aspect, the invention provides a cell for use in a method of therapy, wherein the cell comprises an immune-shielded lentiviral vector, and wherein the method comprises administering the cell to a minor or pediatric subject.

[0045] In one aspect, the present invention provides the use of an immunoshielded lentiviral vector in the preparation of a medicament for treating or preventing a disease, wherein the treatment or prevention comprises administering the immunoshielded lentiviral vector to a minor or pediatric subject. In one aspect, the present invention provides the use of a cell in the preparation of a medicament for treating or preventing a disease, wherein the cell comprises an immunoshielded lentiviral vector, and wherein the treatment or prevention comprises administering the cell to a minor or pediatric subject.

[0046] In one aspect, the invention provides a method of treatment, wherein the method comprises administering an immunoshielded lentiviral vector to a minor or pediatric subject. In one aspect, the invention provides a method of treatment, wherein the method comprises administering a cell to a minor or pediatric subject, wherein the cell comprises an immunoshielded lentiviral vector.

[0047] In one aspect, the invention provides an immune-shielded lentiviral vector for use in a method of treating a minor or pediatric subject. In one aspect, the invention provides a cell for use in a method of treating a minor or pediatric subject, wherein the cell comprises an immune-shielded lentiviral vector.

[0048] In one aspect, the present invention provides the use of an immune-shielded lentiviral vector in the preparation of a medicament for treating or preventing a disease in a minor or pediatric subject. In one aspect, the present invention provides the use of a cell in the preparation of a medicament for treating or preventing a disease in a minor or pediatric subject, wherein the cell comprises an immune-shielded lentiviral vector.

[0049] In one aspect, the present invention provides a method of treating a minor or pediatric subject, wherein the method comprises administering an immune-shielded lentiviral vector to a minor or pediatric subject in need thereof. In one aspect, the present invention provides a method of treating a minor or pediatric subject, wherein the method comprises administering a cell to a minor or pediatric subject in need thereof, wherein the cell comprises an immune-shielded lentiviral vector.

[0050] In some embodiments, the subject is a neonatal subject or an infant subject.

[0051] In some embodiments, the method comprises transducing the hepatocytes with an immune-shielded lentiviral vector. In some embodiments, the method comprises transducing the hepatocytes with an immune-shielded lentiviral vector.

[0052] In some embodiments, the therapy, treatment, or prevention is treatment or prevention of methylmalonic acidemia (MMA).

[0053] In some embodiments, the immune-shielded lentiviral vector comprises a nucleotide sequence encoding a methylmalonic acidemia (MMA)-associated polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 - Design and testing of lentiviral vectors (LV) encoding the MUT transgene

[0055] (a) Schematic representation of the third-generation lentiviral vector (provirus). LTR, long terminal repeat: 5'LTR and 3'LTR each have an almost complete deletion of the U3 region (ΔU3); ψ, psi packaging sequence; RRE, Rev response element; cPPT, central polypurine tract; ET, enhanced transthyretin promoter; MUT wt / co, MUT coding sequence (wild type or codon-optimized); WPRE, posttranscriptional element from the woodchuck hepatitis virus genome; 4x mir142T, four copies of the miR-142 target sequence in tandem. (b) Western blot analysis of MUT in Huh7 cells transduced with LV-ET.MUTwt or LV-ET.MUTco. kDa, molecular weight marker; control, untransduced cells. (c) Western blot analysis of MUT in Huh7 cells with LV-hAAT.MUTwt or LV-hAAT.MUTco. kDa, molecular weight marker. (d) MUT protein expression in Huh7 transduced with LV-ET.MUTwt / co at different MOIs, as determined by WB densitometry analysis and normalized to vector copy number (VCN). (e) MUT protein expression in Huh7 transduced with LV-hAAT.MUTwt / co at different MOIs, as determined by WB densitometry analysis and normalized to vector copy number (VCN). (f) MUT protein expression in Huh7 cells transduced with LV-ET.BSEPco_1 or LV-hAAT.BSEPco_1, as determined by WB densitometry analysis and normalized to the GAPDH housekeeping gene.

[0056] Figure 2 - In vivo gene transfer using LV-ET.MUT

[0057] (a) Vector copy number per cell (VCN / cell) in livers of C57Bl / 6 mice treated with LV-ET.MUTwt or LV-ET.MUTco. (b) MUT mRNA, measured as WPRE normalized to HPRT expression in mice treated with LV-ET.MUTwt or LV-ET.MUTco. (c) Western blot analysis of MUT in liver samples from C57Bl / 6 mice treated with LV-ET.MUTwt or LV-ET.MUTco. (d) MUT protein expression determined by WB densitometric analysis and normalized to the β-actin housekeeping gene in Huh7 cells transduced with LV-ET.MUTco or LV-hAAT.MUTco. UT, untreated mice.

[0058] Figure 3 - Evaluation of long-term LV gene therapy in MCK-MUT- / - mice

[0059] (a) Percent MCK-Mut survival over time. [Mantel-Cox test p=0.0033]. (b) Measurement of MCK-Mut weight (grams) over time. ****p<000.1 by two-way ANOVA comparing MCK-Mut- / -+LV vs. MCK-Mut- / - groups. (c) Measurement of blood methylmalonic acid (MMA) in MCK-Mut mice over time. Student t-test of 50-week post-LV data, p=0.00029 MCK-Mut- / -+LV vs. MCK-Mut- / -. (d) Measurement of serum FGF-21 biomarker. Multiple comparisons of experimental groups were performed by one-way ANOVA. MCK-Mut- / -, untreated KO; MCK-Mut- / -+LV, KO animals treated with LV-ET.MUTwt; MCK-Mut+ / -, heterozygous control animals (phenotypically normal).

[0060] Figure 4 -Vector copy number and MUT protein expression in liver MCK-Mut- / - treated mice 12 months after LV administration

[0061] (a) Vector copy number per cell (VCN / cell) assessed in the liver, (b) MUT mRNA, measured as WPRE normalized on HPRT expression, and (c) Western blot analysis of MUT in liver samples from MCK-Mut mice 12 months after LV administration. KO+LV, KO animals treated with LV-ET.MUT; WT, phenotypically normal animals, KO, untreated KO (MCK-Mut- / -).

[0062] Figure 5 - Transduction efficiency and histopathology of livers of MCK-Mut- / - treated mice 6 months after LV administration (a) RNA in situ hybridization on liver tissue sections with a probe against the WPRE element. (b) Image quantification of liver transduction area in MCK-Mut- / - mice treated with LV-ET.MUT. (c) Liver histopathology images. KO+LV, KO animals treated with LV-ET.MUT; WT, phenotypically normal animals, KO, untreated KO (MCK-Mut- / -).

[0063] Figure 6 -Amelioration of hepatic mitochondrial disease in MCK-Mut- / - mice

[0064] (a) Example electron microscopy images of liver samples. (bd) Image quantification of (b) mitochondrial area, (c) mitochondrial perimeter and (d) mitochondrial shape measured as roundness index [two-way ANOVA, p<0.0001****]. (e) Quantification of methylmalonic acid (liver MMA) in liver tissue. [one-way ANOVA, p<0.0001****]. KO+LV, KO animals treated with LV-ET.MUT; WT, phenotypically normal animals, KO, untreated KO (MCK-Mut- / -).

[0065] Figure 7 -Amelioration of renal mitochondrial disease in MCK-Mut- / - mice

[0066] (a) Example electron microscopy images of kidney samples. (b) Image quantification of ring structures [two-way ANOVA, p<0.0001****]. (c) Quantification of methylmalonic acid (kidney MMA) in kidney tissue. [Kruskal-Wallis multiple comparison test, p=0.04*]. KO UT, untreated KO (MCK-Mut- / -); KO+LV, KO animals treated with LV-ET.MUT; WT, phenotypically normal animals.

[0067] Figure 8 - Reduction of brain MMA in MCK-Mut- / -treated mice

[0068] (a) Example electron microscopy images of brain samples. Quantification of methylmalonic acid (brain MMA) in the brain. [Kruskal-Wallis multiple comparison test, p=0.02*]. KO UT, untreated KO (MCK-Mut- / -); KO+LV, KO animals treated with LV-ET.MUT; WT, phenotypically normal animals.

[0069] Fig. 9 - Using CD47 高 / MHC 无 In vivo gene therapy of LV.MUTwt

[0070] (a) Measurement of MCK-Mut weight (g) over time. (b) Measurement of blood methylmalonic acid (MMA) in MCK-Mut mice over time. KO UT, untreated KO (MCK-Mut- / -); KO+LV, CD47 高 / MHC-I 无 LV.MUTwt-treated KO animals; WT, phenotypically normal animals.

[0071] Fig.10- Comparison of in vivo gene therapy with LV.MUTwt and LV.MUTco at a matched LV dose of 2.5E10 TU / kg

[0072] (a) Measurement of MCK-Mut weight (g) over time. (b) Measurement of blood methylmalonic acid (MMA) in MCK-Mut mice over time. KO UT, untreated KO (MCK-Mut- / -); KO+LV.MUT, KO animals treated with LV-ET.MUTwt; KO+LV.MUTco, KO animals treated with LV-ET.MUTco; WT, phenotypically normal animals.

[0073] Fig.11 - Design and testing of lentiviral vectors (LV) encoding the BSEP transgene

[0074] (a) Schematic representation of the third-generation lentiviral vector (provirus). LTR, long terminal repeat: 5'LTR and 3'LTR each have an almost complete deletion of the U3 region (ΔU3); ψ, psi packaging sequence; RRE, Rev response element; cPPT, central polypurine tract; ET, enhanced transthyretin promoter; BSEP wt / co, BSEP coding sequence (wild type or codon-optimized); WPRE, posttranscriptional element from the woodchuck hepatitis virus genome; 4x mir142T, four copies of the miR-142 target sequence in tandem. (b) Flow cytometric analysis of Huh7 cells transduced with LV-ET.BSEPwt, LV-ET.BSEPco_1, or LV-ET.BSEPco_2 at different MOIs. UT, untransduced Huh7 cells. MFI, mean fluorescence intensity. (c) Histogram reporting the vector copy number (VCN) per cell (VCN / cell) in Huh7 transduced with LV-ET.BSEPwt, LV-ET.BSEPco_1 or LV-ET.BSEPco_2 at different MOIs. (d) Histogram reporting the vector copy number (VCN) per cell (VCN / cell) in Huh7 transduced with LV-ET.BSEPco_1 or LV-hAAT.BSEPco_1 at different MOIs. (e) Western blot analysis of BSEP protein expression performed on Huh7 cell lysates. UT, untransduced Huh7; hAAT, cells transduced with LV-hAAT.BSEPco; ET, cells transduced with LV-ET. (f) BSEP protein expression in Huh7 cells transduced with LV-ET.BSEPco or LV-hAAT.BSEPco as determined by WB densitometry analysis and normalized to the GAPDH housekeeping gene.

[0075] Fig.12- Immunofluorescence analysis of LV-ET.BSEPco_1 in transduced Huh7 cells

[0076] Upper left panel, Huh7 cells transduced with LV-ET.BSEPco_1 and stained with Hoechst. Upper right panel, Huh7 cells not transduced (Cntrl-) and stained with Hoechst. Lower left panel, Huh7 cells transduced with LV-ET.BSEPco_1 and stained with anti-BSEP antibody. Lower right panel, Huh7 cells not transduced (Cntrl-) and stained with anti-BSEP antibody.

[0077] Fig.13 - Evaluation of LV gene therapy in Abcb11- / - mice

[0078] (a) Serum bile acids in Abcb11- / - animals treated with LV-ET.BSEPco (KO+LV) compared to Abcb11- / - untreated animals (KO UT) and WT untreated animals (WT UT). (b) Serum total bilirubin in Abcb11- / - animals treated with LV-ET.BSEPco (KO+LV) compared to Abcb11- / - untreated animals (KOUT). The dashed line indicates the upper limit of bilirubin levels (mg / dL) measured in WT control mice. (c) Serum alkaline phosphatase (ALP) levels (IU / L) in Abcb11- / - untreated animals (KO UT) and WT untreated animals (WT UT). The dashed line indicates the upper limit of ALP levels measured in the WT control mouse cohort.

[0079] Fig.14 - Design and testing of lentiviral vectors (LV) encoding the BSEP transgene

[0080] (a) Schematic diagram of the third-generation lentiviral vector (provirus). LTR, long terminal repeat: 5'LTR and 3'LTR each have an almost complete deletion of the U3 region (ΔU3); ψ, psi packaging sequence; RRE, Rev response element; cPPT, central polypurine tract; ET, enhanced transthyretin promoter; DBT wt / co, DBT coding sequence (wild type or codon optimized); WPRE, posttranscriptional element from the woodchuck hepatitis virus genome; 4x mir142T, four copies of the miR-142 target sequence in tandem. (b) Western blot analysis of DBT in Huh7 cells transduced with LV-ET-DBTwt or LV-ET-DBTco. kDa, molecular weight marker; control, untransduced cells. (c) Western blot analysis of DBT in Huh7 cells transduced with LV-hAAT-DBTwt or LV-hAAT-DBTco. kDa, molecular weight marker; control, untransduced cells. (d) DBT protein expression in Huh7 transduced with LV-ET.DBTwt / co at different MOIs, as determined by WB densitometry analysis and normalized to vector copy number (VCN). (e) VCN in Huh7 transduced with LV-hAAT.DBTwt / co at different MOIs. (f) DBT protein expression in Huh7 cells transduced with LV-ET.DBTwt / co or LV-hAAT.DBTwt / co, as determined by WB densitometry analysis and normalized to the GAPDH housekeeping gene.

[0081] Fig.15 - In vivo gene transfer using LV-ET.DBT

[0082] (a) Vector copy number (VCN) in liver of C57Bl / 6WT mice LV-administered with LV-ET.DBTwt or LV-ET.DBTco. (b) DBT mRNA normalized to HPRT expression in mice treated with LV-ET.DBTwt or LV-ET.DBTco. UT, untreated mice. (c) Western blot analysis of DBT in liver samples from C57Bl / 6 mice treated with LV-ET.DBTwt or LV-ET.DBTco. (d) DBT protein expression as determined by WB densitometry analysis and normalized to the β-actin housekeeping gene.

[0083] Fig.16 - Evaluation of LV gene therapy in iMSUD mice

[0084] (a) Survival of iMSUD mice treated with LV-ET.DBTco (KO+LV) compared to iMSUD untreated (KO UT) and WT control littermates (WT). ****p<0.0001, long rank test. (b) Branched chain amino acid levels normalized to alanine (BCAA / Ala) in iMSUD mice treated with LV-ET.DBTco (KO+LV) compared to iMSUD untreated (KO UT) and WT control littermates (WT). (c) Alloisoleucine (μM) in iMSUD mice treated with LV-ET.DBTco (KO+LV) compared to iMSUD untreated (KO UT) and WT control littermates (WT). DETAILED DESCRIPTION

[0085] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0086] As used herein, the terms "comprising, comprises and comprised of" are synonymous with "including and includes" or "containing and contains" and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps. The terms "comprising, comprises and comprised of" also include the term "consisting of".

[0087] Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation, respectively.

[0088] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0089] Those skilled in the art will understand that they can combine all features of the present invention disclosed herein without departing from the scope of the invention disclosed.

[0090] Lentiviral vectors

[0091] In one aspect, the present invention provides a lentiviral vector comprising a protein coding sequence, wherein the protein coding sequence is a nucleotide sequence encoding a methylmalonic acidemia (MMA)-associated polypeptide.

[0092] "Lentiviral vector" may refer to an enveloped lentiviral genome (i.e., lentiviral particles). For example, the pharmaceutical composition of the present invention preferably comprises a lentiviral vector in the form of lentiviral particles, and the lentiviral vector is preferably administered in the form of lentiviral particles. In other embodiments, a "lentiviral vector" may comprise a lentiviral genome, optionally wherein the lentiviral genome is enveloped. As used herein, a "lentiviral genome" may refer to a genome comprising at least one element derived from or derivable from a lentiviral genome. Suitably, the lentiviral genome comprises at least one element involved in the following mechanism, by which the lentivirus infects cells, expresses genes, and / or replicates.

[0093] Lentivirus is a genus of retroviruses that contains an RNA genome that is converted to DNA in transduced cells by a virally encoded reverse transcriptase. Lentivirus vectors can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, which results in long-term expression of protein coding sequences both in vitro and in vivo (see, e.g., Tiscornia, G., et al., 2006. Nature protocols, 1(1), pp. 241-245).

[0094] The essential genes required for the survival and function of lentiviruses are gag, pol, and env genes: gag encodes structural proteins; pol encodes enzymes required for reverse transcription and integration into the host cell genome; and env encodes viral envelope glycoproteins (see, e.g., Milone, MC and O'Doherty, U., 2018. Leukemia, 32(7), pp. 1529-1541). Lentiviruses may also have additional cis-acting elements, such as rev response elements (RREs), which enable efficient export of RNA transcripts of the integrated provirus from the nucleus of infected target cells to the cytoplasm; retroviral psi packaging elements, which are involved in regulating the essential process of packaging the retroviral RNA genome into the viral capsid during replication; primer binding sites (PBS), where reverse transcription is initiated; TAT activation regions (TARs); splice donor and acceptor sites; and central and terminal polypurine tracts that allow initiation of positive strand synthesis.

[0095] In the lentiviral genome, these elements are usually flanked by regions called long terminal repeats (LTRs) at both ends. LTRs are responsible for integration and transcription. LTRs can also act as enhancer-promoter sequences and can control the expression of lentiviral genes. LTRs themselves are identical or nearly identical sequences, which can generally be divided into three regions: U3, R, and U5. LTRs may be naturally occurring or may be modified. For example, U3 and U5 modifications are described in Iwakuma, T., et al., 1999. Virology, 261 (1), pp. 120-132.

[0096] The lentiviral vector of the present invention may include a minimal lentiviral genome. As used herein, "minimal lentiviral genome" may mean that the lentiviral genome has been engineered to remove non-essential elements and retain essential elements to provide infection, transduction, and the functions required for delivering the nucleotide sequence of interest to the target host cell (see, e.g., Kim, VN, et al., 1998. Journal of virology, 72 (1), pp. 811-816 and Sertkaya, H., et al., 2021. Scientific reports, 11 (1), pp. 1-15).

[0097] The lentiviral genome may comprise from 5' to 3': 5'LTR, one or more lentiviral derived cis-acting elements and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, RRE and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, retroviral psi packaging element, RRE and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, retroviral psi packaging element, RRE, cPPT and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, PBS, retroviral psi packaging element, RRE, cPPT and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, PBS, retroviral psi packaging element, RRE, cPPT and 3'LTR.

[0098] The lentiviral genome may further comprise a protein coding sequence and optionally one or more regulatory elements (e.g., operably linked to a protein coding sequence). Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, RRE, protein coding sequence, and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, retroviral psi packaging element, RRE, protein coding sequence, and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, retroviral psi packaging element, RRE, cPPT, protein coding sequence, and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, retroviral psi packaging element, RRE, cPPT, protein coding sequence, and 3'LTR. Suitably, the lentiviral genome may comprise from 5' to 3': 5'LTR, PBS, retroviral psi packaging element, RRE, cPPT, protein coding sequence, and 3'LTR.

[0099] The lentiviral vector of the present invention may be replication-deficient. Typically, at least a portion of one or more protein coding regions essential for replication may be removed from the lentiviral genome. This makes the lentiviral vector "replication-deficient" or "replication-incompetent". Suitably, one or more of the gag, pol, rev and env genes are deleted (at least partially) in the replication-deficient lentiviral vector. Suitably, each of the gag, pol, rev and env genes is deleted (at least partially) in the replication-deficient lentiviral vector. Optionally, the lentiviral vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0100] The lentiviral vector of the present invention can be derived from any lentivirus. As used herein, "lentiviral derived" or "lentiviral based" can mean that the lentiviral genome contains one or more elements from the lentivirus. For example, the coding region of the viral protein can be deleted, but one or more cis-acting elements can be retained from the lentivirus.

[0101] Lentiviral vectors can be derived from primate lentiviruses. Examples of "primate" lentiviruses include, but are not limited to, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Lentiviral vectors can be derived from non-primate lentiviruses (i.e., derived from lentiviruses that do not primarily infect primates, especially humans). Examples of "non-primate" lentiviruses include, but are not limited to, the prototype "lentivirus" visna / maedi virus (VMV), caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[0102] Suitably, the lentiviral vector of the present invention is a HIV-derived lentiviral vector. As used herein, "HIV-derived" or "HIV-based" may mean that the lentiviral genome includes one or more elements from HIV. For example, the coding region of HIV viral proteins may be missing, and one or more HIV cis-acting elements may be retained in the lentiviral genome (see, e.g., Johnson, NM, et al., 2021. Molecular Therapy-Methods & Clinical Development, 21, pp. 451-465). The HIV-derived lentiviral genome may include from 5' to 3': 5'LTR, one or more HIV-derived cis-acting elements (e.g., RRE and / or cPPT) and 3'LTR.

[0103] Suitably, the lentiviral vector of the present invention is a lentiviral vector derived from HIV-1. The prototype lentiviral vector system is based on HIV-1 (see, for example, Merten, OW, et al., 2016.Molecular Therapy-Methods & Clinical Development, 3, p.16017). It has been shown that sequences extending into the gag open reading frame can be important for packaging HIV-1. Therefore, HIV-1 vectors generally contain relevant parts of gag in which the translation start codon has been mutated. In addition, HIV-1 vectors generally also contain a part of the env gene including RRE. Rev binds to RRE, and RRE allows full-length or single-spliced ​​mRNA to be transported from the nucleus to the cytoplasm. In the absence of rev and / or RRE, full-length HIV-1 RNA can be accumulated in the nucleus. Alternatively, constitutive transport elements from some simple retroviruses such as Mason-Pfizer monkey viruses can be used to alleviate the demand for rev and RRE. The HIV-1 derived lentiviral genome may comprise, from 5' to 3': a 5' LTR, one or more HIV-1 derived cis-acting elements (eg, PBS, retroviral psi packaging element, RRE and / or cPPT), and a 3' LTR.

[0104] The lentiviral vector of the present invention can be a self-inactivating lentiviral vector. As used herein, a "self-inactivating" or "SIN" lentiviral vector can include a lentiviral genome in which lentiviral enhancer and promoter sequences have been deleted (see, for example, Zufferey, R., et al., 1998. Journal of virology, 72 (12), pp. 9873-9880 and Miyoshi, H., et al., 1998. Journal of virology, 72 (10), pp. 8150-8157). SIN lentiviral vectors can be generated and transduced in vivo with an efficacy similar to that of wild-type vectors. The transcriptional inactivation of the long terminal repeats (LTR) in the SIN provirus can prevent the mobilization of replication-competent viruses. This can also achieve regulated expression of genes from internal promoters by eliminating any cis-acting effects of LTR.

[0105] The lentiviral vectors of the present invention may be integration-competent. As used herein, an "integration-competent" lentiviral vector is capable of integrating into the genome of a host cell. In contrast to integration-competent lentiviral vectors, integration-defective lentiviral vectors may be generated, for example, by packaging a lentiviral vector with a catalytically inactive integrase (such as an HIV integrase carrying a D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the lentiviral genome LTR, or by a combination of the above (see, e.g., Wanisch, K. and RJ, 2009. Molecular Therapy, 17(8), pp.1316-1332).

[0106] The lentiviral vectors of the present invention may be replication-defective and integrative. The lentiviral vectors of the present invention may be replication-defective, integrative and self-inactivating. The lentiviral vectors of the present invention may be replication-defective, integrative, self-inactivating and HIV-derived.

[0107] The lentiviral vector of the present invention can be a lentiviral particle. "Lentiviral particle" can refer to an enveloped lentiviral genome. Lentiviral particles can be produced by co-transfecting a plasmid containing a lentiviral genome (e.g., a "transfer vector") with a helper plasmid (e.g., a "packaging vector" encoding gag-pol and / or rev and an "envelope vector" encoding env) into a host cell and then harvesting the supernatant containing the lentivirus.

[0108] The lentiviral vector of the present invention may be pseudotyped. Pseudotyping lentiviral vectors with naturally occurring or engineered lentiviral envelopes can allow targeted transduction of specific cell types (see, e.g., Joglekar, AV and Sandoval, S., 2017. Human Gene Therapy Methods, 28(6), pp. 291-301). Suitably, pseudotyped lentiviral vectors are used to allow transduction of liver cells (e.g., hepatocytes).

[0109] The lentiviral vector of the present invention can be VSV-G pseudotyped. Vesicular stomatitis virus G protein (VSV-G) is a commonly used envelope protein for pseudotyping. VSV-G is a trimeric protein that binds to phosphatidylserine and low-density lipoprotein receptors on the cell surface to be internalized into the cell. VSV-G pseudotyped lentiviral vectors can effectively transduce liver cells (e.g., hepatocytes).

[0110] The lentiviral vectors of the invention may be replication-defective, integrative, and VSV-G pseudotyped. The lentiviral vectors of the invention may be replication-defective, integrative, self-inactivating, and VSV-G pseudotyped. The lentiviral vectors of the invention may be replication-defective, integrative self-inactivating, HIV-derived, and VSV-G pseudotyped.

[0111] Immune shielded lentiviral vectors

[0112] The lentiviral vector of the present invention may be an immune-shielded lentiviral vector.

[0113] As used herein, "immunoshielded lentiviral vector" may refer to a lentiviral vector modified to reduce an immune response after administration. For example, immune shielding may reduce the activation of an acute inflammatory response after administration.

[0114] In some embodiments, the lentiviral vector of the invention is: (i) CD47 高 Lentiviral vector; (ii) MHC-I 无 and / or (iii) comprising one or more miRNA target sequences (e.g., which inhibit transgene expression in antigen presenting cells). In a preferred embodiment, the lentiviral vector of the present invention is a CD47 高 / MHC-I 无 The lentiviral vector comprises one or more miRNA target sequences (eg, which inhibit transgene expression in antigen presenting cells). Each of these modifications can act to reduce the immune response following administration.

[0115] The immune-shielded lentiviral vector can be "phagocytosis-shielded" to reduce uptake by professional phagocytes. In some embodiments, the lentiviral vector is a phagocytosis-shielded lentiviral vector. For example, the lentiviral vector can be a CD47 高 Lentiviral vectors.

[0116] CD47 高 Lentiviral vectors

[0117] The lentiviral vector of the present invention may be CD47 高 Lentiviral vector. As used herein, "CD47 高 A "lentiviral vector" may refer to a lentiviral vector having increased levels of CD47 (or a fragment thereof) on its surface. 高 Lentiviral vectors may have reduced uptake by professional phagocytes.

[0118] CD47 (cluster of differentiation 47), also known as integrin-associated protein protein (IAP), is a transmembrane protein encoded by the CD47 gene in humans. Phagocytosis is physiologically inhibited by CD47, which is a ubiquitously expressed ligand for the signal regulatory protein alpha (SIRP-α) receptor, which is expressed by professional phagocytes. CD47 can be incorporated into lentiviral vectors when they bud from producer cells.

[0119] The lentiviral vectors of the present invention may comprise one or more CD47 polypeptides (or fragments thereof) on their surface. The amount of CD47 (or fragments thereof) on the surface may be sufficient to reduce uptake by professional phagocytes. Any suitable assay may be used to quantify the amount of CD47 polypeptides (or fragments thereof) present on the surface of the lentiviral vector.

[0120] In some embodiments, the density of CD47 polypeptide (or fragment thereof) can be determined by immunostaining of CD47 and total internal reflection fluorescence microscopy, for example as described in US 2010 / 0316570A1. The density of CD47 polypeptide (or fragment thereof) can be determined by immunostaining of CD47 and total internal reflection fluorescence microscopy, for example as described in US 2010 / 0316570A1. 2 , at least about 25 molecules / μm 2 , at least about 30 molecules / μm 2 , at least about 35 molecules / μm 2 , at least about 40 molecules / μm 2 , at least about 45 molecules / μm 2 , at least about 50 molecules / μm 2 , at least about 60 molecules / μm 2 , at least about 70 molecules / μm 2 , at least about 80 molecules / μm 2, at least about 90 molecules / μm 2 , at least about 100 molecules / μm 2 , at least about 150 molecules / μm 2 , at least about 200 molecules / μm 2 , at least about 250 molecules / μm 2 , at least about 300 molecules / μm 2 , at least about 350 molecules / μm 2 , at least about 400 molecules / μm 2 , at least about 450 molecules / μm 2 , at least about 500 molecules / μm 2 , at least about 600 molecules / μm 2 , at least about 700 molecules / μm 2 , at least about 800 molecules / μm 2 , at least about 900 molecules / μm 2 or at least about 1000 molecules / μm 2 CD47 polypeptide (or its fragment) can be present at a density of about 1000 molecules / μm 2 or less, about 500 molecules / μm 2 or less, or about 250 molecules / μm 2 CD47 polypeptide (or its fragment) may be present at a density of about 20 molecules / μm 2 About 1000 molecules / μm 2 , about 20 molecules / μm 2 About 500 molecules / μm 2 or about 20 molecules / μm 2 to about 250 molecules / μm 2 The density exists.

[0121] In some embodiments, the amount of CD47 polypeptide (or fragment thereof) can be determined by immunostaining for CD47 and electron microscopy, as described in Milani, M., et al., 2019. Science Translational Medicine, 11 (493), p.eaav732. The CD47 polypeptide (or fragment thereof) can be detected in an amount of at least about 10 gold particles / lentiviral particle, at least about 15 gold particles / lentiviral particle, or at least about 20 gold particles / lentiviral particle. The CD47 polypeptide (or fragment thereof) can be detected in an amount of about 100 gold particles / lentiviral particle or less, about 80 gold particles / lentiviral particle or less, or about 60 gold particles / lentiviral particle or less. The CD47 polypeptide (or fragment thereof) can be detected in an amount of about 10 to about 100 gold particles / lentiviral particle, about 15 to about 80 gold particles / lentiviral particle, or about 20 to about 60 gold particles / lentiviral particle.

[0122] The lentiviral vector of the present invention can be obtained from CD47 高 Host cell. As used herein, "CD47 高 A "host cell" may refer to a host cell having increased levels of CD47 (or a fragment thereof) on its surface.

[0123] CD47 高 The host cell is genetically engineered to increase the expression of CD47 (or a fragment thereof) on the cell surface. For example, the host cell may contain a vector encoding CD47 (or a fragment thereof), or may be edited to introduce a nucleotide sequence encoding CD47 (or a fragment thereof) into its genome. Suitably, the host cell is transduced with a viral vector encoding a CD47 polypeptide (or a fragment thereof).

[0124] CD47 高 The host cell may have a higher concentration of CD47 (or a fragment thereof) on its surface than an unmodified host cell (e.g., an unmodified producer cell or packaging cell as described herein). Suitably, the host cell has at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold more CD47 on its cell surface than an unmodified host cell. Suitably, the host cell has about 5-fold to about 30-fold more CD47 (or a fragment thereof) on its cell surface than an unmodified host cell.

[0125] Suitably, the lentiviral vector of the invention has a higher concentration of CD47 (or a fragment thereof) on its surface than a lentiviral vector obtained from an unmodified host cell (e.g., an unmodified producer cell or packaging cell as described herein). Suitably, the lentiviral vector has at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times more CD47 (or a fragment thereof) on its surface than a lentiviral vector obtained from an unmodified host cell. Suitably, the lentiviral vector has about 5 times to about 30 times more CD47 (or a fragment thereof) on its surface than a lentiviral vector obtained from an unmodified host cell.

[0126] CD47 is a member of the immunoglobulin (Ig) superfamily of membrane proteins with a single IgV-like domain at its N-terminus, which is a highly hydrophobic stretch with five transmembrane segments, and an alternatively spliced ​​cytoplasmic C-terminus ranging from 3 to 36 amino acids in length. Mouse, rat, bovine and human CD47 molecules have been cloned and show about 70% overall amino acid identity (see, e.g., Brown, EJ and Frazier, WA, 2001. Trends in cell biology, 11(3), pp. 130-135).

[0127] The CD47 polypeptide (or its fragment) may be a human CD47 polypeptide (or its fragment). The CD47 polypeptide may have the amino acid sequence of UniProtKB Q08722.

[0128] Exemplary CD47 polypeptides are provided by SEQ ID NOs: 1-4. Suitably, the CD47 polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NOs: 1-4. Suitably, the CD47 polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0129] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRNN

[0130] Exemplary CD47 polypeptide (SEQ ID NO:1)

[0131] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE

[0132] Exemplary CD47 polypeptide (SEQ ID NO:2)

[0133] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIV IFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFV

[0134] Exemplary CD47 polypeptide (SEQ ID NO: 3)

[0135] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILL FWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGGLLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLN

[0136] Exemplary CD47 polypeptide (SEQ ID NO:4)

[0137] SEQ ID NOs: 5-8 provide exemplary CD47 polypeptides that do not include a signal peptide. Suitably, the CD47 polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NOs: 5-8. Suitably, the CD47 polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 5-8.

[0138] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRNN

[0139] Exemplary CD47 polypeptide without a signal peptide (SEQ ID NO: 5)

[0140] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE

[0141] Exemplary CD47 polypeptide without a signal peptide (SEQ ID NO: 6)

[0142] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILL FWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGGLLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFV

[0143] Exemplary CD47 polypeptide not comprising a signal peptide (SEQ ID NO: 7)

[0144] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKT LKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLN

[0145] Exemplary CD47 polypeptide not comprising a signal peptide (SEQ ID NO: 8)

[0146] Those skilled in the art will be able to generate variants and / or fragments based on conservative substitutions and / or known structural and functional characteristics of CD47. These are described, for example, in Fenalti, G., et al., 2021. Nature communications, 12(1), pp. 1-14.

[0147] Suitably, fragments of CD47 and / or CD47 variants retain the ability to inhibit phagocytosis. Suitably, CD47 fragments and / or CD47 variants may comprise the extracellular domain of CD47. The extracellular domain of human CD47 may interact with SIRP-α and inhibit phagocytosis. Optionally, CD47 fragments and / or CD47 variants comprise the transmembrane domain of CD47. The domains may be connected by interdomain linkers. Fragments and / or variants may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the activity of a full-length CD47 polypeptide.

[0148] Suitably, the variant of SEQ ID NO: 1 may comprise one or more variations selected from the group consisting of: V5I, C14W, C15R, F22L, S27F, F30L, F32Y, T36S, V38L, V38I, F42V, T44A, N50S, T51A, T52S, T52A, V56I, R63K, A71T, S75Y, T76A, P78L, P78S, P78A, S82R, S82N, S83T, K85N, K85E, V88A, V88L, V88I, Q90R, L91F, K93N, M100I, M10 0V, D101G, K102R, K102T, S107L, I126F, I127V, K130Q, R132H, S138F, V146I, I150V, I153V, S169A, G170R, G170S, G171S, D173Y, I177V, A178G, V181I, V185A, I186V, V18 8A, I189T, I191V, V198I, A207S, T215I, I219M, Y226C, A231S, T235A, S236F, A240V, A240T, I241V, V243I, I244T, V246L, Y249F, A252S, A252T, V254A, S257T, I264M, I264L, M266I, M266T, M266V, V287I, V292A, N295S, N295D, Q296L, P302S, N304S, and N304D. These were considered tolerable, benign, and / or likely benign variants as predicted by SIFT, PolyPhen, CADD, REVEL, and MetaLR.

[0149] Suitably, the variant of SEQ ID NO: 2 may comprise one or more variations selected from the group consisting of P3L, A6P, F22L, S27F, F30L, F32Y, T36S, V38L, V38I, F42V, N50S, T51A, T52S, T52A, V56I, R63K, A71T, S75Y, T76A, P78L, P78S, P78A, S82R, S82N, S83T, K85N, K85E, V88A, V88L, V88I, Q90R, L91F, K93N, M100I, M100V, D101G, K102R, K102T, S107L, I126F, I127V, K130Q, R132H, S133 38F, V146I, I150V, I153V, S169A, G170R, G170S, G171S, I177V, A178G, V181 I, I186V, V188A, I189T, I191V, V198I, A207S, T215I, I219M, Y226C, A231S, T 235A, A240V, A240T, I241V, V243I, I244T, V246L, Y249F, A252S, A252T, V254A, I264M, I264L, M266I, M266T, M266V, V287I, V292A, N295S, N295D, and Q296L. These were considered tolerable, benign, and / or likely benign variants as predicted by SIFT, PolyPhen, CADD, REVEL, and MetaLR.

[0150] An exemplary CD47 fragment is provided by SEQ ID NO: 9. Suitably, the CD47 fragment comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 9. Suitably, the CD47 fragment comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0151] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPN

[0152] Exemplary CD47 fragment (SEQ ID NO:9)

[0153] An exemplary CD47 fragment that does not include a signal peptide is provided by SEQ ID NO: 10. Suitably, the CD47 fragment comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 10. Suitably, the CD47 fragment comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0154] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPN

[0155] Exemplary CD47 fragment not comprising a signal peptide (SEQ ID NO: 10)

[0156] MHC-I 低 MHC-I 无 Lentiviral vectors

[0157] The lentiviral vector of the present invention may be MHC-I 低 Lentiviral vector or MHC-I 无 In a preferred embodiment, the lentiviral vector of the present invention is an MHC-I 无 Lentiviral vectors.

[0158] As used herein, "MHC-I 低 A "lentiviral vector" may refer to a lentiviral vector having reduced levels of one or more MHC-I molecules on its surface (i.e., reduced levels of surface-exposed MHC-I molecules). The number of surface-exposed MHC-I molecules may be reduced such that the immune response to MHC-I is reduced to a therapeutically relevant level.

[0159] As used herein, "MHC-I 无 A "lentiviral vector" may refer to a lentiviral vector that substantially lacks (or is free of) one or more MHC-I molecules on its surface (i.e., substantially lacks (or is free of) surface-exposed MHC-I molecules).

[0160] Major histocompatibility complex class I (MHC-I) is a heterodimeric membrane protein displayed on the outer leaflet of the cell membrane (see, e.g., Penn, DJ and Ilmonen, P., 2005. Major histocompatibility complex (MHC). eLS). MHC-I functions to bind peptide fragments of proteins and display them to the extracellular environment, where they can be recognized by CD8+ cytotoxic T cells. Due to central and peripheral tolerance mechanisms, peptide fragments generated by normal cell proteins will not activate cytotoxic T cells. However, exogenous peptides (e.g., those derived from viral proteins) will cause activation of immune responses to destroy cells. Allogeneic MHC-I proteins themselves can be recognized by the immune system. For example, antibodies can directly bind to MHC-I epitopes. Therefore, lentiviral vectors containing MHC-I molecules derived from allogeneic sources can be targeted and neutralized by the immune system.

[0161] The term "MHC-I molecule" may refer to a human MHC-I molecule. Human MHC-I is also known as human leukocyte antigen class I (HLA-I) and is expressed on almost all nucleated cells. HLA-I consists of two polypeptide chains, HLA-I heavy chain (α chain) and β2 microglobulin (β2M or β chain). The HLA-I α chain and β2M are non-covalently linked. The HLA-I α chain is polymorphic. To date, six HLA-I α chains have been identified, including three classical, highly polymorphic α chains (HLA-A, HLA-B and HLA-C) and three non-classical, less polymorphic (HLA-E, HLA-F and HLA-G) α chains. The MHC-I molecule may comprise or consist of HLA-A, HLA-B and HLA-C molecules, which contain an invariant β2M sequence.

[0162] The term "MHC-I molecule" may also include variant MHC-I sequences, such as polymorphisms of HLA-1 α chain sequences and / or β2M sequences. For example, a variant MHC-I sequence may include an HLA-1 α chain sequence and / or β2M sequence having a single nucleotide polymorphism (SNP) or multiple SNPs.

[0163] Any suitable assay can be used to quantify the amount of MHC-I molecules present on the surface of a lentiviral vector.

[0164] In some embodiments, the amount of MHC-I molecules can be determined by immunostaining and electron microscopy for MHC-I, as described in Milani, M., et al., 2017. EMBO molecular medicine, 9 (11), pp. 1558-1573. MHC-I molecules can be detected in an amount of less than about 10 gold particles / lentiviral particles, less than about 9 gold particles / lentiviral particles, less than about 8 gold particles / lentiviral particles, less than about 7 gold particles / lentiviral particles, less than about 6 gold particles / lentiviral particles, less than about 5 gold particles / lentiviral particles, less than about 4 gold particles / lentiviral particles, less than about 3 gold particles / lentiviral particles, less than about 2 gold particles / lentiviral particles, less than about 1 gold particle / lentiviral particle, or about 0 gold particles / lentiviral particles. MHC-I molecules can be undetectable (e.g., the amount of gold particles detected can be not significantly higher than the background level).

[0165] The lentiviral vector of the present invention can be obtained from MHC-I 低 Host cell or MHC-I 无 In a preferred embodiment, the lentiviral vector of the present invention is obtained from MHC-I 无 Host cell. As used herein, "MHC-I 低 A "host cell" may refer to a host cell having reduced levels of one or more MHC-I molecules on its surface. As used herein, "MHC-I 无 A "host cell" may refer to a host cell that is substantially lacking or absent one or more MHC-I molecules on its surface.

[0166] MHC-I can be genetically engineered 低 MHC-I 无 The host cell can be engineered to reduce expression of MHC-I on the cell surface. For example, the cell can comprise a genetically engineered disruption of a gene encoding β2-microglobulin and / or a genetically engineered disruption of a gene encoding an MHC-I α chain.

[0167] Methods for reducing the genetic engineering of protein expression are known in the art. For example, this can be achieved by targeted gene knockout. In order to reduce protein expression, the gene encoding the protein itself or its regulatory sequence (such as its promoter) can be knocked out. Knockout can be achieved by deleting a section of the coding nucleic acid sequence (it can be missing a section of the protein necessary for expression or stability) or changing the reading frame of the coding sequence or by base editing. Suitable methods for targeted gene knockout include using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases based on CRISPR / Cas (see, for example, Gaj, T. et al., 2013. Trends Biotechnol, 31, pp. 397-405). For example, if there is provided with a suitable RNA guide designed to bind to a specific locus, the nuclease guided by CRISPR / Cas9 RNA can be used to catalyze the double-stranded break at the locus in the genome. Cas9 and guide RNA can be delivered to target cells by transfecting a vector encoding protein and RNA. The cell attempts to repair any double-strand breaks in its DNA using the non-homologous end joining (NHEJ) pathway. This is an error-prone mechanism that inserts random nucleotides and often disrupts the reading frame of the target gene. Alternatively, genetic engineering to reduce protein expression can be achieved using RNAi technology, microRNAs, or antisense RNAs to inhibit the expression of the target gene.

[0168] Once targeted gene knockout or expression inhibition methods have been performed, the resulting cell populations can be screened to select and enrich for cells that exhibit a phenotype of interest (e.g., reduced expression of surface-exposed MHC-I). Suitable techniques for screening and enrichment are known in the art and include flow cytometry and fluorescence-activated cell sorting (FACS).

[0169] In some embodiments, the host cell comprises a genetically engineered disruption of a gene encoding β2-microglobulin. β2-microglobulin stabilizes MHC-I, so cells lacking β2-microglobulin will exhibit reduced expression of MHC-I on the cell surface. The cell may comprise a genetically engineered disruption in all copies of the gene encoding β2-microglobulin.

[0170] In another embodiment, the cell comprises a genetically engineered disruption of one or more genes encoding an MHC-I alpha chain.The cell may comprise a genetically engineered disruption in all copies of a gene encoding an MHC-I alpha chain.

[0171] The cell may contain both an engineered disruption of the gene encoding β2-microglobulin and an engineered disruption of the gene encoding the MHC-I α chain.

[0172] Reduced expression of MHC-I on the surface of a cell can refer to a reduction in the number of MHC-I molecules expressed on the surface of a genetically engineered cell compared to the number of MHC-I molecules expressed on the surface of a cell in the absence of genetic engineering but otherwise substantially the same conditions. The expression of MHC-I on the surface of a cell can be reduced such that the number of surface-exposed MHC-I molecules is, for example, less than about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the number of surface-exposed MHC-I molecules displayed in the absence of genetic engineering. In some embodiments, the expression of MHC-I on the surface of a cell is reduced such that the number of surface-exposed MHC-I molecules is 0% of the number of surface-exposed MHC-I molecules displayed in the absence of genetic engineering.

[0173] Preferably, the expression of MHC-I on the cell surface is reduced such that the cell is substantially devoid of surface-exposed MHC-I molecules. In this context, "substantially devoid" can mean that there is a significant reduction in the number of MHC-I molecules expressed on the surface of the genetically engineered cell compared to the number of MHC-I molecules expressed on the surface of the cell lacking the genetic engineering, such that the immune response to MHC-I on the lentiviral vector generated by the cell is reduced to a therapeutically useful extent.

[0174] Suitably, the lentiviral vector of the invention has a lower concentration of MHC-I molecules on its surface than a lentiviral vector obtained from an unmodified host cell (e.g., an unmodified production cell or packaging cell as described herein). Suitably, the lentiviral vector has less than about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the number of surface-exposed MHC-I molecules displayed on a lentiviral vector obtained from an unmodified host cell. In some embodiments, the lentiviral vector has less than about 20% of the number of surface-exposed MHC-I molecules displayed on a lentiviral vector obtained from an unmodified host cell.

[0175] In some embodiments, the lentiviral vector of the invention substantially lacks MHC-I molecules on its surface. In this context, "substantially lacking" can mean that there is no detectable immune response due to the molecules on the surface of the lentiviral vector.

[0176] In some embodiments, the lentiviral vector of the present invention has no MHC-I molecules on its surface. In this context, "no" can mean that there are no detectable molecules (e.g., by immunostaining and electron microscopy) on the surface of the lentiviral vector. As used herein, "undetectable" can refer to a level that is not statistically significantly different from the background level.

[0177] In some embodiments, the lentiviral vector of the invention has reduced HLA-A, HLA-B and / or HLA-C molecules on its surface. Suitably, the lentiviral vector has less than about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the number of surface-exposed HLA-A molecules displayed on the lentiviral vector obtained from an unmodified host cell. Suitably, the lentiviral vector has less than about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the number of surface-exposed HLA-B molecules displayed on the lentiviral vector obtained from an unmodified host cell. Suitably, the lentiviral vector has less than about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the number of surface exposed HLA-C molecules displayed on the lentiviral vector obtained from an unmodified host cell.

[0178] In some embodiments, the lentiviral vector of the present invention substantially lacks HLA-A, HLA-B and / or HLA-C molecules on its surface. In some embodiments, the lentiviral vector of the present invention substantially lacks HLA-A, HLA-B and HLA-C molecules on its surface. In some embodiments, the lentiviral vector of the present invention has no HLA-A, HLA-B and / or HLA-C molecules on its surface. In some embodiments, the lentiviral vector of the present invention has no HLA-A, HLA-B and / or HLA-C molecules on its surface.

[0179] As mentioned above, the HLA-I molecule is composed of two polypeptide chains, the HLA-I heavy chain (α chain) and β2 microglobulin (β2M or β chain). The HLA-I α chain and β2M are non-covalently linked.

[0180] A skilled person will be able to readily determine the amino acid and nucleic acid sequence of the HLA-1 alpha chain. For example, in a genomic sequence, the location of the HLA-I alpha chain within the major histocompatibility complex region of the chromosome can be used to identify the HLA-I alpha chain (see, e.g., Penn, DJ and Ilmonen, P., 2005. Major histocompatibility complex (MHC). eLS).

[0181] The HLA-A alpha chain may have the amino acid sequence of UniProtKB P04439. Exemplary HLA-A alpha chains are provided by SEQ ID NOs: 11 and 12. Suitably, the HLA-A alpha chain comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NOs: 11 or 12. Suitably, the HLA-A alpha chain comprises or consists of the amino acid sequence of SEQ ID NOs: 11 or 12.

[0182] MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHEAEQLRA YLDGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKV

[0183] Exemplary HLA-A alpha chain (SEQ ID NO: 11)

[0184] MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLE GRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSYTQAASSDSAQGSDVSLTACKV

[0185] Exemplary HLA-A alpha chain (SEQ ID NO: 12)

[0186] The HLA-B alpha chain may have the amino acid sequence of UniProtKB P01889. SEQ ID NO: 13 provides an exemplary HLA-B alpha chain. Suitably, the HLA-B alpha chain comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 13. Suitably, the HLA-B alpha chain comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0187] MLVMAPRTVLLLLSAALALTETWAGSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRR AYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSYSQAACSDSAQGSDVSLTA

[0188] Exemplary HLA-B alpha chain (SEQ ID NO: 13)

[0189] The HLA-Cα chain may have the amino acid sequence of UniProtKB P10321. Exemplary HLA-Cα chains are provided by SEQ ID NOs: 14 and 15. Suitably, the HLA-Cα chain comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NOs: 14 or 15. Suitably, the HLA-Cα chain comprises or consists of the amino acid sequence of SEQ ID NOs: 14 or 15.

[0190] MRVMAPRALLLLLSGGLALTETWACSHSMRYFDTAVSRPGRGEPFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAY LEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEPSSQPTIPIMGIVAGLAVLVVLAVLGAVVTAMMCRRKSSGGKGGSCSQAACSNSAQGSDESLITCKA

[0191] Exemplary HLA-Cα chain (SEQ ID NO: 14)

[0192] MRVMAPRALLLLLSGGLALTETWACSHSMRYFDTAVSRPGRGEPFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNEHLRSCTAADTA AQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLRWGGKGGSCSQAACSNSAQGSDESLITCKA

[0193] Exemplary HLA-Cα chain (SEQ ID NO: 15)

[0194] The amino acid and nucleic acid sequences encoding β2M are also known in the art. For example, the nucleic acid sequence of human β2M is deposited as GenBank Accession No. NM_004048.

[0195] The HLA β chain can be the HLA β chain of UniProtKB P61769. SEQ ID NO: 16 provides an exemplary HLA β chain. Suitably, the HLA β chain comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identity to SEQ ID NO: 16. Suitably, the HLA β chain comprises or consists of the amino acid sequence of SEQ ID NO: 16.

[0196] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0197] Exemplary HLA β chain (SEQ ID NO: 16)

[0198] The lentiviral vector of the present invention may be CD47 高 / MHC-I 无 Lentiviral vector or CD47 高 / MHC-I 低 In a preferred embodiment, the lentiviral vector of the present invention is CD47 高 / MHC-I 无 Lentiviral vectors.

[0199] The lentiviral vector of the present invention can be obtained from CD47 高 / MHC-I 无 Host cell or CD47 高 / MHC-I 低 In a preferred embodiment, the lentiviral vector of the present invention is obtained from a CD47 高 / MHC-I 无 Host cell.

[0200] miRNA target sequences

[0201] The lentiviral vector of the present invention may contain one or more miRNA target sequences. One or more miRNA target sequences may be operably linked to a protein coding sequence. The term "operably linked" may mean that the components are in a relationship that allows them to function in their intended manner.

[0202] MicroRNA (miRNA) genes are scattered in all human chromosomes except the Y chromosome. Similar to protein-coding genes, miRNAs are usually transcribed from polymerase II promoters, which generate so-called primary miRNA transcripts (pri-miRNAs). A stem loop of about 60 nucleotides in length is excised from pri-miRNA, called miRNA precursor (pre-miRNA), leaving 5' phosphate and 2bp long 3' overhangs. The pre-miRNA is then actively transported from the nucleus to the cytoplasm. Then, Dicer performs double-stranded cleavage at the other end of the stem loop to generate a 19-24bp duplex, which is composed of mature miRNA and the opposite strand of the duplex, called miRNA*. One strand of the duplex is selectively loaded into the RNA-induced silencing complex (RISC) and stacked as a mature microRNA. This chain is usually a chain that is not too tightly paired with its complement at its 5' end. However, there are some miRNAs that support the stacking of two duplex chains to a similar degree.

[0203] Once loaded into RISC, the guide strand of the mature microRNA interacts with mRNA target sequences preferentially found in the 3' untranslated region (3'UTR) of protein-coding genes. If the entire guide strand sequence is perfectly complementary to the mRNA target, the mRNA is cleaved by an endonuclease. If only the seed sequence (i.e., nucleotides 2-8 counted from the 5' end of the miRNA) is perfectly complementary to the target mRNA, RNAi can work through an alternative mechanism that leads to translational inhibition.

[0204] Protein expression from a protein coding sequence (i.e., "transgene expression") can be regulated by one or more endogenous miRNAs using one or more corresponding miRNA target sequences. Using this approach, one or more miRNAs endogenously expressed in a cell prevent or reduce transgene expression in the cell by interacting with its corresponding miRNA target sequence located in the lentiviral genome (see, e.g., Brown, BD et al. (2007) Nat Biotechnol 25: 1457-1467).

[0205] Suitable miRNA target sequences for inhibiting transgenic expression in specific cells are known to the skilled person. Techniques known to those skilled in the art can be used to determine miRNAs with a desired expression profile. For example, mammalian microRNA expression atlases are described in Landgraf, P., et al., 2007. Cell, 129 (7), pp. 1401-1414, and the distribution of miRNA expression in human tissues is described in Ludwig, N., et al., 2016. Nucleic acids research, 44 (8), pp. 3865-3877. Once a miRNA has been identified, the corresponding target sequence can be easily determined using, for example, a microRNA database such as miRBase (Griffiths-Jones, S., et al., 2007. Nucleic acids research, 36 (suppl_1), pp. D154-D158).

[0206] The miRNA target sequence can be completely or partially complementary to the corresponding miRNA. As used herein, the term "completely complementary" can mean that the target sequence has a nucleic acid sequence that is 100% complementary to the miRNA sequence that recognizes it. As used herein, the term "partially complementary" can mean that the target sequence is only partially complementary to the sequence of the miRNA that recognizes it, so that the partially complementary sequence is still recognized by the miRNA. In other words, in the context of the present invention, the partially complementary target sequence effectively recognizes the corresponding miRNA in the cell expressing the miRNA and effectively prevents or reduces transgenic expression. Suitably, the partially complementary miRNA target sequence can be completely complementary to the miRNA seed sequence.

[0207] Including more than one copy of the miRNA target sequence in the lentiviral vector can increase the effectiveness of the system. In addition, different miRNA target sequences can be included. For example, the protein coding sequence can be operably linked to more than one miRNA target sequence, and the miRNA target sequences can be different or the same. The miRNA target sequences can be tandem, but other arrangements are envisioned. The lentiviral vector can, for example, contain 1, 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequences. Suitably, the lentiviral vector contains 4 miRNA target sequences for each miRNA target sequence.

[0208] The copies of the miRNA target sequence may be separated by a spacer sequence. The spacer sequence may comprise, for example, at least one, at least two, at least three, at least four, or at least five nucleotide bases.

[0209] Suitably, the lentiviral vector comprises one or more miRNA target sequences, two or more miRNA target sequences, three or more miRNA target sequences, or four or more miRNA target sequences. Suitably, the protein coding sequence is operably linked to one or more miRNA target sequences, two or more miRNA target sequences, three or more miRNA target sequences, or four or more miRNA target sequences. In some embodiments, the protein coding sequence is operably linked to four miRNA target sequences.

[0210] The miRNA target sequence may be a human miRNA target sequence. Suitably, the miRNA target sequence is a -5p or -3p miRNA target sequence.

[0211] The one or more miRNA target sequences can inhibit transgene expression in one or more cells other than liver cells (eg, hepatocytes).

[0212] One or more miRNA target sequences can inhibit transgene expression in hematopoietic lineage cells. Hematopoietic stem cells lead to different types of blood cells, which are called myeloid and lymphoid in the system. As used herein, "hematopoietic lineage cells" can include myeloid cells and lymphoid cells. Myeloid cells can include monocytes, macrophages, neutrophils, basophils and eosinophils. Lymphoid cells can include T cells, B cells, natural killer cells and innate lymphoid cells.

[0213] One or more miRNA target sequences can inhibit transgene expression in antigen presenting cells. As used herein, "antigen presenting cells" (APC) can refer to cells that present antigens bound by major histocompatibility complex (MHC) proteins on their surface. APC can be a hematopoietic lineage cell. Antigen presenting cells can be professional antigen presenting cells. Professional APCs present antigens specifically to T cells and can include macrophages, B cells and dendritic cells. Suitably, APCs are spleen and / or liver APCs.

[0214] One or more miRNA target sequences can inhibit transgene expression in antigen presenting cells of the hematopoietic lineage.

[0215] By preventing transgene expression in antigen-presenting cells while allowing high-level expression in other cells, miRNA regulation can achieve strong and stable gene transfer in the absence of an immune response.

[0216] As used herein, the term "inhibit expression" can refer to a reduction in expression in a relevant cell type of a transgene operably linked to one or more miRNA target sequences compared to transgene expression in the absence of one or more miRNA target sequences but under otherwise substantially identical conditions. In some embodiments, transgene expression is inhibited by at least 50%. In some embodiments, transgene expression is inhibited by at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, transgene expression is substantially prevented, e.g., undetectable.

[0217] The miRNA-mediated method for limiting gene expression has several advantages relative to other strategies for regulating transgenic. Although expression can be successfully restricted to target cells using tissue-specific promoters, leaky expression is observed in a portion of non-target cells. This occurs because the reconstructed promoter modified to be included in the vector system usually loses some of its cell specificity, and also because the vector integration near the active promoter and enhancer may activate the tissue-specific promoter and drive transgenic expression. In contrast, because the silencing mediated by miRNA occurs at the post-transcriptional level, promoter and enhancer capture are unrelated. Therefore, miRNA regulation can be used to effectively de-target transgenic expression from specific cell types, while still allowing extensive tissue expression. MiRNA regulation can also be used in combination with tissue-specific promoter / enhancer. By including the miRNA target sequence in the expression cassette already under the control of the tissue-specific promoter, an extra regulatory layer that can eliminate off-target expression has been added.

[0218] Exemplary miRNA target sequences for inhibiting transgenic expression in hematopoietic lineage cells and / or antigen presenting cells include, but are not limited to, miR-181, miR-142, miR-223, and miR-155 target sequences. Other miRNA target sequences for inhibiting transgenic expression in hematopoietic lineage cells and / or antigen presenting cells are known in the art (see, for example, Ghafouri-Fard, S., et al., 2021. Non-coding RNA research, 6 (1), pp. 8-14). MiRNAs expressed in hematopoietic lineage cells and / or antigen presenting cells interact with corresponding miRNA target sequences and reduce the expression of target genes (see, for example, Brown, BD, et al., 2006. Nature medicine, 12 (5), pp. 585-591 and Brown, BD, et al., 2007. Nature biotechnology, 25 (12), pp. 1457-1467).

[0219] Further miRNA target sequences that inhibit transgene expression in cells of the hematopoietic lineage and / or antigen presenting cells can be identified by any suitable method, such as miRNA expression analysis as described in Monticelli, S., et al., 2005. Genome biology, 6(8), pp. 1-15.

[0220] Suitably, the one or more miRNA target sequences comprise or consist of: (i) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8) miR-142 target sequences; (ii) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8) miR-181 target sequences; (iii) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8) miR-223 target sequences; and / or (iv) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8) miR-155 target sequences.

[0221] In some embodiments, one or more miRNA target sequences comprise or consist of: (i) two or more miR-142 target sequences; (ii) two or more miR-181 target sequences; (iii) two or more miR-223 target sequences; and / or (iv) two or more miR-155 target sequences. In some embodiments, one or more miRNA target sequences comprise or consist of: (i) at least four miR-142 target sequences; (ii) at least four miR-181 target sequences; (iii) at least four miR-223 target sequences; and / or (iv) at least four miR-155 target sequences. In some embodiments, one or more miRNA target sequences comprise or consist of: (i) four miR-142 target sequences; (ii) four miR-181 target sequences; (iii) four miR-223 target sequences; and / or (iv) four miR-155 target sequences. Suitably, the target sequences are separated by spacer sequences.

[0222] In some embodiments, one or more miRNA target sequences comprise or consist of: one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8) miR-142 target sequences. In some embodiments, one or more miRNA target sequences comprise or consist of: two or more miR-142 target sequences. In some embodiments, one or more miRNA target sequences comprise or consist of: three or more miR-142 target sequences. In some embodiments, one or more miRNA target sequences comprise or consist of: four or more miR-142 target sequences. In some embodiments, one or more miRNA target sequences comprise or consist of: four miR-142 target sequences. Suitably, the target sequences are separated by spacer sequences.

[0223] The miR-142 target sequence can be a human miRNA target sequence. Suitably, the miR-142 target sequence is a miR-142-5p or miR-142-3p miRNA target sequence. In some embodiments, the miR-142 target sequence is a miR-142-3p miRNA target sequence.

[0224] In some embodiments, the miR-142 target sequence comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO: 17, or a fragment thereof. Suitably, the miR-142 target sequence comprises or consists of an amino acid sequence at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 17, or a fragment thereof.

[0225] In some embodiments, the miR-142 target sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 17 or a fragment thereof.

[0226] TCCATAAAGTAGGAAACACTACA

[0227] Exemplary miR-142 target sequence (SEQ ID NO: 17)

[0228] In some embodiments, the one or more miRNA target sequences comprise or consist of an amino acid sequence at least 70% identical to SEQ ID NO: 18, or a fragment thereof. Suitably, the one or more miRNA target sequences comprise or consist of a nucleotide sequence at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 18, or a fragment thereof.

[0229] In some embodiments, the one or more miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 18 or a fragment thereof.

[0230] TCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACAACCGGTTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACA

[0231] Exemplary 4x miR-142 target sequence (SEQ ID NO: 18)

[0232] MMA-associated peptides

[0233] The protein coding sequence delivered by the lentiviral vector of the present invention is a nucleotide sequence encoding a methylmalonic acidemia (MMA) associated polypeptide. Once delivered to a cell, the protein encoded by the protein coding sequence can be expressed in the cell (ie, "transgenic expression" can occur).

[0234] As used herein, "MMA-associated polypeptides" may be any polypeptide associated with methylmalonic acidemia (MMA). As described above, MMA is a group of inborn errors of metabolism associated with elevated methylmalonic acid concentrations in blood and urine, caused by the failure of methylmalonyl-CoA to be converted to succinyl-CoA during propionyl-CoA metabolism in the mitochondrial matrix (see, e.g., Manoli I, et al. 2005. Isolated Methylmalonic Acidemia. In Gene Reviews). MMA-associated polypeptides may be any polypeptide associated with the pathway of methylmalonyl-CoA conversion to succinyl-CoA, including polypeptides associated with intracellular cobalamin metabolism (e.g., polypeptides encoded by MMUT, MMAA, MMAB, MMACHC, MMADHC, LMBRD1, ABCD4, HCFC1, MCEE, SUCLA2 / SUCLG1, ACSF3 or ALDH6A1).

[0235] In a preferred embodiment, the MMA-associated polypeptide is an iMMA-associated polypeptide. As described above, isolated MMA (iMMA) may be caused by the following: the enzyme methylmalonyl-CoA mutase (encoded by MMUT) is completely (mut 0 Enzymatic isoform) or part (mut –defects in the transport or synthesis of its cofactor adenosylcobalamin (caused by pathogenic variants in MMAA, MMAB, or MMADHC); or defects in the enzyme methylmalonyl-CoA epimerase (encoded by MCEE) (see, e.g., Manoli I, et al. 2005. Isolated Methylmalonic Acidemia. In Gene Reviews).

[0236] Suitably, the MMA-associated polypeptide is selected from methylmalonyl-CoA mutase (MMUT) or a fragment and / or variant thereof; methylmalonic aciduria type A (MMAA) or a fragment and / or variant thereof; methylmalonic aciduria type B (MMAB) or a fragment and / or variant thereof; methylmalonic aciduria and homocystinuria type D (MMADHC) or a fragment and / or variant thereof; and methylmalonyl-CoA epimerase (MCEE) or a fragment and / or variant thereof.

[0237] The protein coding sequence can be codon optimized. For example, the protein coding sequence can be codon optimized for expression in mammalian (eg, human) cells.

[0238] Different cells differ in the choice of their specific codons. This codon bias corresponds to the bias in the relative abundance of a specific tRNA in a cell type. By changing the codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. For the same reason, expression can be reduced by deliberately selecting codons that are known to be rare in a specific cell type for the corresponding tRNA. Therefore, an additional degree of translational control can be obtained. For mammalian cells (e.g., humans) and for a variety of other organisms, codon selection tables are known in the art.

[0239] MMUT

[0240] In some embodiments, the MMA-associated polypeptide is MMUT or a fragment and / or variant thereof.

[0241] "MMUT" or "MUT" is an abbreviation for a polypeptide encoded by MMUT, also known as mitochondrial methylmalonyl-CoA mutase (MCM) and methylmalonyl-CoA isomerase. MMUT catalyzes the AdoCbl-dependent rearrangement of methylmalonyl-CoA (mmCoA) to succinyl-CoA (sCoA). Each MMUT monomer is characterized by a dual-domain structure comprising a large substrate-binding TIM barrel connected to a small AdoCbl binding domain via an interdomain linker of about 100 aa, wherein the active site is located at the N / C domain interface (see, e.g., Froese, DS, et al., 2010. Journal of Biological Chemistry, 285 (49), pp. 38204-38213).

[0242] Fragments and / or variants of MMUT may retain MMUT activity (see, e.g., EC 5.4.99.2). For example, fragments and / or variants of MMUT may be able to catalyze the rearrangement of mmCoA to sCoA. Suitably, fragments and / or variants of MMUT may have the same or similar activity as MMUT, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the activity of MMUT. Assays for determining MMUT activity are described in, e.g., Janata, J., et al., 1997. Human Molecular Genetics, 6 (9), pp. 1457-1464 and Forny, P., et al., 2014. Human mutation, 35 (12), pp. 1449-1458.

[0243] "A fragment of MMUT" may refer to a portion or region of a full-length MMUT that has the same or similar activity as the full-length MMUT, i.e., the fragment may be a functional fragment (e.g., the fragment may catalyze the rearrangement of mmCoA to sCoA). The fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the activity of the full-length MMUT.

[0244] "MMUT variants" may comprise an amino acid sequence or nucleotide sequence that is at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to a wild-type MMUT, optionally at least 95% or at least 97% or at least 99% identical. MMUT variants may have the same or similar activity as a wild-type MMUT (e.g., a MMUT variant may catalyze the rearrangement of mmCoA to sCoA). MMUT variants may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity of a wild-type MMUT.

[0245] Based on the known structural and functional characteristics of MMUT (see, for example, Froese, DS, et al., 2010. Journal of Biological Chemistry, 285(49), pp. 38204-38213; NH and Leadlay, PF, 1996. Protein Science, 5(9), pp. 1922-1927; and Acquaviva, C., et al., 2005. Human mutation, 25(2), pp. 167-176) and / or based on known mutations (see, e.g., NCBI Gene ID: 4594 and NCBI Homolo Gene: 20097), a person skilled in the art will be able to use conservative substitutions to generate fragments of MMUT and / or MMUT variants.

[0246] Suitably, the fragment of MMUT and / or MMUT variant comprises an mmCoA binding domain and an AdoCbl binding domain. The fragment of MMUT may also comprise a mitochondrial targeting sequence and / or a dimerization domain. The domains / sequences may be connected via an interdomain linker.

[0247] The MMUT gene is conserved in chimpanzees, rhesus monkeys, dogs, cattle, mice and rats. The MMUT may be a human MMUT. Suitably, the MMUT may comprise or consist of the following: a polypeptide sequence of UniProtKB accession number P22033 or a fragment and / or variant thereof.

[0248] In some embodiments, the MMUT comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 37, or a fragment thereof. Suitably, the MMUT comprises or consists of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 37, or a fragment thereof.

[0249] In some embodiments, the MMUT comprises or consists of the following: SEQ ID NO:37 or a fragment thereof.

[0250] MLRAKNQLFLLSPHYLRQVKESSGSRLIQQRLLHQQQPLHPEWAALAKKQLKGKNPEDLIWHTPEGISIKPLYSKRDTMDLPEELPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTVEESNKFYKDNIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVEDTKILFDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKFNSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVFGGTQSLHTNSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLTNDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDAVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGFADLGFDVDIGPLFQTPREVAQQAVDADVHAVGISTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV

[0251] Exemplary MMUT (SEQ ID NO:37)

[0252] Suitably, the MMUT variant may comprise one or more variations selected from the group consisting of N6K, N6S, Q7R, F9L, S12L, P13L, P13S, H14L, H14R, H14N, Y15N, L16V, R17S, R17K, Q18R, V19G, K20E, E21A, S22L, S22P, L27V, I28L, P38S, A44T, A45S, K48E, K49R, K54R, N55K, D58A, I60 T, I60L, K75R, K75E, R76T, R76K, R76G, T78A, M79T, M79V, M79L, L81I, L85F, L85V, V88M, R154H, R154C, I200 V, P209S, K210E, E211D, E211D, K251N, K251E, H252Y, I274V, S288A, Q293R, H331R, E334K, M336I, M336V, S3 42A, A349T, I372V, S416T, C433W, C433Y, C433R, N436S, N436D, I449V, M452L, E461Q, R475K, A499P, A499T, A504V, A504T, N512K, K519E, K520R, I521V, K522R, S524G, A528V, A528G, E531Q, R532H, E539K, A542V, S543 N, A550V, K571R, K595R, I597L, T598A, T598P, I601V, I601L, H605R, K606R, M608T, R610H, R610C, I634V, I671M, I671V, K687R, S691A, E715K, S719A, N720S, V721I, K730T, V733I, Q734R, D737H, E740K, K741R, and K746T. These were considered tolerable, benign, and / or likely benign variants as predicted by SIFT, PolyPhen, CADD, and EVEL.

[0253] Suitably, the MMUT variant may comprise one or more variants selected from the group consisting of: I69V, R154H, A499T, K520T, K522R, R532G, R532H and I671V. Based on clinical data, these are considered to be benign (or likely benign) variations.

[0254] Suitably, the nucleotide sequence encoding MMUT or a fragment and / or variant thereof may comprise or consist of the nucleotide sequence of NCBI reference sequence NM_000255 or a fragment and / or variant thereof.

[0255] In some embodiments of the present invention, the nucleotide sequence encoding MMUT or a fragment and / or variant thereof comprises or consists of a nucleotide sequence or a fragment thereof that is at least 70% identical to SEQ ID NO: 38. Suitably, the nucleotide sequence encoding MMUT or a fragment and / or variant thereof comprises or consists of a nucleotide sequence or a fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 38.

[0256] In some embodiments of the present invention, the nucleotide sequence encoding MMUT or a fragment and / or variant thereof comprises or consists of the nucleotide sequence of SEQ ID NO: 38 or a fragment thereof.

[0257]

[0258] Exemplary nucleotide encoding MMUT (SEQ ID NO: 38)

[0259] In some embodiments, the nucleotide sequence encoding MMUT or a fragment and / or variant thereof is codon-optimized. An exemplary codon-optimized sequence is provided in SEQ ID NO:39.

[0260] In some embodiments, the nucleotide sequence encoding MMUT, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 70% identical to SEQ ID NO: 39. Suitably, the nucleotide sequence encoding MMUT, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 39.

[0261] In some embodiments, the nucleotide sequence encoding MMUT or a fragment and / or variant thereof comprises or consists of the nucleotide sequence of SEQ ID NO: 39 or a fragment thereof.

[0262]

[0263] Exemplary codon-optimized nucleotide encoding MMUT (SEQ ID NO: 39)

[0264] MMAA

[0265] In some embodiments, the MMA-associated polypeptide is MMAA or a fragment and / or variant thereof.

[0266] "MMAA" is an abbreviation for the polypeptide encoded by the MMAA gene, and is also known as mitochondrial methylmalonic aciduria type A protein and metabolism of cobalamin-associated A. MMAA mediates the transport of cobalamin (Cbl) into mitochondria for the final step of adenosylcobalamin (AdoCbl) synthesis, and assists in the delivery of the AdoCbl cofactor from MMAB to methylmalonyl-CoA mutase (MMUT).

[0267] Fragments and / or variants of MMAA can retain MMAA activity. For example, fragments and / or variants of MMAA can mediate Cbl transport to mitochondria and / or assist AdoCbl cofactor delivery from MMAB to MMUT. Suitably, fragments and / or variants of MMAA can have the same or similar activity as MMAA, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the activity of MMAA. Suitable assays for MMAA activity are known in the art (see, for example, Plessl, T., et al., 2017. Human Mutation, 38 (8), pp. 988-1001).

[0268] Based on the known structural and functional characteristics of MMAA (see, e.g., Plessl, T., et al., 2017. Human Mutation, 38(8), pp. 988-1001 and Froese, DS, et al., 2010. Journal of Biological Chemistry, 285(49), pp. 38204-38213), and / or based on known variants (see, e.g., NCBI Gene ID: 166785 and NCBI HomoloGene: 14586), those skilled in the art will be able to use conservative substitutions to generate fragments and / or variants.

[0269] The MMAA gene is conserved in chimpanzees, dogs, cows, mice and rats. The MMAA may be a human MMAA. Suitably, the MMAA may comprise or consist of the following: a polypeptide sequence of UniProtKB accession number Q8IVH4 or a fragment and / or variant thereof.

[0270] In some embodiments, the MMAA comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 40, or a fragment thereof. Suitably, the MMAA comprises or consists of an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 40, or a fragment thereof.

[0271] In some embodiments, the MMAA comprises or consists of SEQ ID NO: 40 or a fragment thereof.

[0272] MPMLLPHPHQHFLKGLLRAPFRCYHFIFHSSTHLGSGIPCAQPFNSLGLHCTKWMLLSDGLKRKLCVQTTLKDHTEGLSDKEQRFVDKLYTGLIQGQRACLAEA ITLVESTHSRKKELAQVLLQKVLLYHREQEQSNKGKPLAFRVGLSGPPGAGKSTFIEYFGKMLTERGHKLSVLAVDPSSCTSGGSLLGDKTRMTELSRDMNAYIR PSPTRGTLGGVTRTTNEAILLCEGAGYDIILIETVGVGQSEFAVADMVDMFVLLLPPAGGDELQGIKRGIIEMADLVAVTKSDGDLIVPARRIQAEYVSALKLL RKRSQVWKPKVIRISARSGEGISEMWDKMKDFQDLMLASGELTAKRRKQQKVWMWNLIQESVLEHFRTHPTVREQIPLLEQKVLIGALSPGLAADFLLKAFKSRD

[0273] Exemplary MMAA (SEQ ID NO: 40)

[0274] Suitably, the MMAA variant may comprise Q363H. Based on clinical data, this variation is considered benign.

[0275] Suitably, the nucleotide sequence encoding MMAA or a fragment and / or variant thereof may comprise or consist of a nucleotide sequence of NCBI reference sequence NM_172250 or NM_001375644 or a fragment and / or variant thereof.

[0276] In some embodiments, the nucleotide sequence encoding MMAA, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 70% identical to SEQ ID NO: 41. Suitably, the nucleotide sequence encoding MMAA, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 41.

[0277] In some embodiments, the nucleotide sequence encoding MMAA or a fragment and / or variant thereof comprises or consists of the nucleotide sequence of SEQ ID NO: 41 or a fragment thereof.

[0278]

[0279] Exemplary nucleotides encoding MMAA (SEQ ID NO: 41)

[0280] MMAB

[0281] In some embodiments, the MMA-associated polypeptide is MMAB or a fragment and / or variant thereof.

[0282] "MMAB" is an abbreviation for a polypeptide encoded by the MMAB gene, and is also known as mitochondrial methylmalonic aciduria type B protein and corrinoid adenylyltransferase. MMAB catalyzes the final step in the conversion of vitamin B12 to adenosylcobalamin (AdoCbl), and can also deliver AdoCbl to the MMUT.

[0283] Fragments and / or variants of MMAB may retain MMAB activity (see, e.g., EC 2.5.1.17). For example, fragments and / or variants of MMAB may catalyze the final step in the conversion of vitamin B12 to AdoCbl and / or deliver AdoCbl to MMUT. Suitably, fragments and / or variants of MMAB may have the same or similar activity as MMAB, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity of MMAB. Suitable assays for MMAB activity are known in the art (see, e.g., Forny, P., et al., 2021. Humangenetics, pp. 1-15).

[0284] Based on the known structural and functional characteristics of MMAB (see, e.g., Forny, P., et al., 2021. Human genetics, pp. 1-15; and Schubert, HL and Hill, CP, 2006. Biochemistry, 45 (51), pp. 15188-15196), and / or based on known variants (see, e.g., NCBI Gene ID: 326625 and NCBI HomoloGene: 12680), those skilled in the art will be able to use conservative substitutions to generate fragments and / or variants. Suitably, fragments of MMAB and / or MMAB variants comprise a cobalamin adenylyltransferase domain.

[0285] The MMAB gene is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice and rats. The MMAB may be a human MMAB. Suitably, the MMAB may comprise or consist of the following: a polypeptide sequence of UniProtKB accession number Q96EY8 or a fragment and / or variant thereof.

[0286] In some embodiments, the MMAB comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 42, or a fragment thereof. Suitably, the MMAB comprises or consists of an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 42, or a fragment thereof.

[0287] In some embodiments, the MMAB comprises or consists of SEQ ID NO: 42 or a fragment thereof.

[0288] MAVCGLGSRLGLGSRLGLRGCFGAARLLYPRFQSRGPQGVEDGDRPQPSSKTPRIPKIYTKTGDKGFSSTFTGERRPKDDQVFEAVGTTDELSSAIGFALELVTEKGHTFAEELQKIQCTLQDVG SALATPCSSAREAHLKYTTFKAGPILELEQWIDKYTSQLPPLTAFILPSGGKISSALHFCRAVCRRAERRVVPLVQMGETDANVAKFLNRLSDYLFTLARYAAMKEGNQEKIYMKNDPSAESEGL

[0289] Exemplary MMAB (SEQ ID NO: 42)

[0290] Suitably, the MMAB variant may comprise one or more variants selected from the group consisting of C4R, R19H, R19Q, S52N, T62M, S69N, T222M and M239K. Based on clinical data, these are considered to be benign (or likely benign) variants.

[0291] Suitably, the nucleotide sequence encoding MMAB or a fragment and / or variant thereof may comprise or consist of the nucleotide sequence of NCBI reference sequence NM_052845 or a fragment and / or variant thereof.

[0292] In some embodiments, the nucleotide sequence encoding MMAB, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 70% identical to SEQ ID NO: 43. Suitably, the nucleotide sequence encoding MMAB, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 43.

[0293] In some embodiments, the nucleotide sequence encoding MMAB or a fragment and / or variant thereof comprises or consists of the nucleotide sequence of SEQ ID NO: 43 or a fragment thereof.

[0294] ATGGCTGTGTGCGGCCTGGGGAGCCGTCTTGGCCTGGGGAGCCGTCTTGGCCTGCGCGGGTGCTTCGGCGCCGCCAGGCTCCTGTATCCCCGTTTCCAGAGCCGCGGCCCTCAGGGCGTGGAAGACGGGGACAGGCCACAGCCTTCCTCGAAGACACCCAGGATCCCCAAGATTTACACCAAAACGGG AGACAAAGGGTTTTCTAGTACCTTCACAGGAGAAAGGAGACCCAAAGATGACCAAGTGTTTGAAGCCGTGGGAACTACAGATGAATTAAGTTCAGCTATTGGGTTTGCTCTGGAATTAGTCACAGAAAAGGGCCATACATTTGCCGAAGAGCTTCAGAAAATCCAGTGCACATTGCAGGACGTCGGCT CGGCCCTGGCGACACCATGCTCCTCGGCCCGGGAGGCTCACTTAAAGTATACCACGTTCAAGGCGGGCCCATCCTGGAGCTGGAGCAGTGGATCGACAAGTACACCAGCCAGCTCCCACCACTCACGGCCTTCATCCTGCCTTCGGGAGGCAAGATCAGCTCGGCGCTGCATTTCTGCCGGGCCGTG TGCCGCCGGGCCGAGAGACGTGTGGTGCCTCTTGTCCAGATGGGAGAGACCGATGCGAACGTGGCCAAGTTCTTAAACAGACTCAGTGACTATCTCTTCACGCTAGCCAGATATGCAGCCATGAAGGAGGGGAATCAAGAGAAAATATACATGAAAAATGACCCATCGGCCGAGTCTGAGGGACTCTGA

[0295] Exemplary nucleotides encoding MMAB (SEQ ID NO: 43)

[0296] MMADHC

[0297] In some embodiments, the MMA-associated polypeptide is MMADHC or a fragment and / or variant thereof.

[0298] "MMADHC" is the abbreviation of the polypeptide encoded by the MMADHC gene, and is also called the cobalamin transport protein CblD and mitochondrial methylmalonic aciduria and homocystinuria type D protein. MMADHC participates in cobalamin metabolism and transport, and plays an important role in processing Cbl cofactors and targeting Cbl cofactors to their target enzymes. It has been proposed that Cbl bound to MMADHC can form a partner with MMADHC to ferry to methylmalonyl-CoA mutase (see, e.g., Froese, DS, et al., 2015. Journal of Biological Chemistry, 290 (49), pp. 29167-29177).

[0299] The fragments and / or variants of MMADHC can retain the MMADHC activity. For example, the fragments and / or variants of MMADHC can transport Cbl to methylmalonyl-CoA mutase. Suitably, the fragments and / or variants of MMADHC can have the same or similar activity as MMADHC, for example, can have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the activity of MMADHC.

[0300] Based on the known structural and functional characteristics of MMADHC (see, e.g., Froese, DS, et al., 2015. Journal of Biological Chemistry, 290(49), pp. 29167-29177; Jusufi, J., et al., 2014. Journal of inherited metabolic disease, 37(5), pp. 841-849; Deme, JC, et al., 2012. Molecular genetics and metabolism, 107(3), pp. 352-362; and Yamada, K., et al., 2015. Journal of Biological Chemistry, 290(49), pp. 29155-29166) and / or based on known variants (see, e.g., NCBI Gene ID: 27249 and NCBI HomoloGene: 9248), those skilled in the art will be able to use conservative substitutions to generate fragments and / or variants.

[0301] The MMADHC gene is conserved in chimpanzees, rhesus monkeys, dogs, cattle, mice and rats. The MMADHC may be human MMADHC. Suitably, the MMADHC may comprise or consist of the following: a polypeptide sequence of UniProtKB accession number Q9H3L0 or a fragment and / or variant thereof.

[0302] In some embodiments, the MMADHC comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 44, or a fragment thereof. Suitably, the MMADHC comprises or consists of an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 44, or a fragment thereof.

[0303] In some embodiments, the MMADHC comprises or consists of SEQ ID NO: 44 or a fragment thereof.

[0304] MANVLCNRARLVSYLPGFCSLVKRVVNPKAFSTAGSSGSDESHVAAAAPPDICSRTVWPDETMGPFGPQDQRFQLPGNIGFDCHLNGTASQKKSLVHKTLPDVLAEPLSSERHEFVMAQYVNEFQGNDAPVEQEINSAETYFESARVEC AIQTCPELLRKDFESLFPEVANGKLMILTVTQKTKNDMTVWSEEVEIEREVLLEKFINGAKEICYALRAEGYWADFIDPSSGLAFFGPYTNNTLFETDERYRHLGFSVDDLGCCKVIRHSLWGTHVVVGSIFTNATPDSHIMKKLSGN

[0305] Exemplary MMADHC (SEQ ID NO: 44)

[0306] Suitably, the MMADHC variant may comprise one or more variants selected from the group consisting of K29N, E138K, S143I, R158Q, N206S and P236L. Based on clinical data, these are considered to be benign (or likely to be benign) variations.

[0307] Suitably, the nucleotide sequence encoding MMADHC or a fragment and / or variant thereof may comprise or consist of the nucleotide sequence of NCBI reference sequence NM_015702 or a fragment and / or variant thereof.

[0308] In some embodiments, the nucleotide sequence encoding MMADHC, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 70% identical to SEQ ID NO: 45. Suitably, the nucleotide sequence encoding MMADHC, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 45.

[0309] In some embodiments, the nucleotide sequence encoding MMADHC or a fragment and / or variant thereof comprises or consists of the nucleotide sequence of SEQ ID NO: 45 or a fragment thereof.

[0310] ATGGCCAATGTGCTTTGTAACAGAGCCAGACTGGTTTCCTATCTCCCAGGATTTTGCTCTTTAGTTAAAAGGGTTGTCAATCCCAAAGCCTTTTCGACTGCAGGATCATCAGGTTCGGATGAGTCTCATGTGGCTGCTGCACCTCCAGATATATGCTCTCGAACAGTGTGGCCTGATGAAACTATGGGACCCTTTGGACCTCAAGATCAGAGGTTCCAGCTTCCTGGGAACATAGGTTTTGATTGTCACCTCAATGGGACTGCTTCACAGAAGAAAAGCCTGGTTCATAAAACTTTGCCTGATGTTCTAGCAGAACCTTTATCAAGTGAAAGACATGAGTTTGTGATGGCACAATATGTGAATGAATTTCAGGGTAATGATGCACCTGTTGAACAAGAAATTAACAGTGCAGAAACTTACTTTGAAAGTGCCAGAGTAGAGTGTGCAATACAGACATGTCCAGAATTGCTGCGAAAAGATTTTGAATCACTGTTTCCAGAAGTAGCTAATGGCAAACTAATGATTCTGACTGTAACACAAAAAACTAAGAATGATATGACTGTTTGGAGTGAAGAAGTAGAAATTGAAAGAGAAGTGCTCTTAGAAAAGTTCATCAATGGTGCTAAGGAAATTTGCTATGCTCTTCGAGCTGAGGGTTATTGGGCTGACTTTATTGACCCATCATCTGGTTTGGCATTTTTTGGACCATATACAAACAACACTCTTTTTGAAACTGATGAACGCTACCGACATTTAGGATTCTCTGTTGATGACCTTGGATGCTGTAAAGTGATTCGTCATAGTCTCTGGGGTACCCATGTAGTTGTAGGGAGTATCTTCACTAATGCAACACCAGACAGCCATATTATGAAGAAATTAAGTGGAAATTAG

[0311] Exemplary nucleotide encoding MMADHC (SEQ ID NO:45)

[0312] MCEE

[0313] In some embodiments, the MMA-associated polypeptide is MCEE or a fragment and / or variant thereof.

[0314] "MCEE" is an abbreviation for the polypeptide encoded by the MCEE gene, and is also known as mitochondrial methylmalonyl-CoA epimerase and methylmalonyl-CoA racemase. MCEE interconverts D-methylmalonyl-CoA and L-methylmalonyl-CoA during the degradation of branched-chain amino acids, odd-chain fatty acids, and other metabolites.

[0315] Fragments and / or variants of MCEE may retain MCEE activity (see, e.g., EC 5.1.99.1). For example, fragments and / or variants of MCEE may convert (S)-methylmalonyl-CoA to (R)-methylmalonyl-CoA. Suitably, fragments and / or variants of MCEE may have the same or similar activity as MCEE, for example, may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the activity of MCEE. Suitable assays for MCEE activity are known in the art (see, e.g., K., et al., 2019. BBA-Molecular Basis of Disease, 1865 (6), pp. 1265-1272).

[0316] Based on the known structural and functional characteristics of MCEE (see, e.g., Heuberger, K., et al., 2019. BBA-Molecular Basis of Disease, 1865(6), pp. 1265-1272), and / or based on known variants (see, e.g., NCBI Gene ID: 84693 and NCBI HomoloGene: 13078), a person skilled in the art will be able to use conservative substitutions to generate fragments and / or variants.

[0317] The MCEE gene is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice and rats. MCEE may be human MCEE. Suitably, MCEE may comprise or consist of the following: the polypeptide sequence of UniProtKB accession number Q96PE7 or a fragment and / or variant thereof.

[0318] In some embodiments, MCEE comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 46, or a fragment thereof. Suitably, MCEE comprises or consists of an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 46, or a fragment thereof.

[0319] In some embodiments, MCEE comprises or consists of SEQ ID NO: 46 or a fragment thereof.

[0320] MARVLKAAAANAVGLFSRLQAPIPTVRASSTSQPLDQVTGSVWNLGRLNHVAIAVPDLEKAAAFYKNILGAQVSEAVPLPEHGVSVVFVNLGNTKMELLHPLGRDSPIAGFLQKNKAGGMHHICIEVDNINAAVMDLKKKKIRSLSEEVKIGAHGKPVIFLHPKDCGGVLVELEQA

[0321] Exemplary MCEE (SEQ ID NO: 46)

[0322] Suitably, the MCEE variant may comprise one or more variants selected from the group consisting of: A76V, R104L and R143H. Based on clinical data, these are considered to be benign (or likely benign) variations.

[0323] Suitably, the nucleotide sequence encoding MCEE or a fragment and / or variant thereof may comprise or consist of the nucleotide sequence of the NCBI reference sequence NM_032601 or a fragment and / or variant thereof.

[0324] In some embodiments, the nucleotide sequence encoding MCEE, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 70% identical to SEQ ID NO: 47. Suitably, the nucleotide sequence encoding MCEE, or a fragment and / or variant thereof, comprises or consists of a nucleotide sequence or a fragment thereof that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 47.

[0325] In some embodiments, the nucleotide sequence encoding MCEE or a fragment and / or variant thereof comprises or consists of the nucleotide sequence of SEQ ID NO: 47 or a fragment thereof.

[0326] ATGGCGCGGGTGCTGAAGGCTGCAGCCGCGAATGCCGTAGGGCTTTTTTCCAGACTTCAAGTCCCCATTCCAACAGTAAGAGCTTCTTCCACATCACAGCCCTTGGATCAAGTGACAGGTTCTGTGTGGAAC CTGGGTCGACTCAACCATGTAGCCATAGCAGTGCCAGATTTGGAAAAGGCTGCAGCATTTTATAAGAATATTCTGGGGGCCCAGGTAAGTGAAGCGGTCCCTCTTCCTGAACATGGAGTATCTGTTGTTTTTG TCAACCTGGGAAATACCAAGATGGAACTGCTTCATCCATTGGGACGTGACAGTCCAATTGCAGGTTTTCTGCAGAAAAACAAGGCTGGAGGAATGCATCACATCTGCATCGAGGTGGATAATATTAATGCAGC TGTGATGGATTTGAAAAAAAAAGAAGATCCGCAGTCTAAGTGAAGAGGTCAAAATAGGAGCACATGGAAAACCAGTGATTTTTCTCCATCCTAAAGACTGTGGTGGAGTCCTTGTGGAACTGGAGCAAGCTTGA

[0327] Exemplary nucleotide encoding MCEE (SEQ ID NO: 47)

[0328] Regulatory elements

[0329] The lentiviral vector of the present invention may further comprise one or more regulatory elements that can act before or after transcription. Suitably, the protein coding sequence is operably linked to one or more regulatory elements that can act before or after transcription. One or more regulatory elements can promote the expression of transgenes in liver cells (e.g., hepatocytes).

[0330] As used herein, "regulatory element" may refer to any nucleotide sequence that promotes polypeptide expression, such as acting to increase transcript expression or enhance mRNA stability. Suitable regulatory elements include, for example, promoters, enhancer elements, post-transcriptional regulatory elements, polyadenylation sites and Kozak sequences.

[0331] Promoter

[0332] The lentiviral vector of the present invention may comprise a promoter, preferably a liver-specific (eg, hepatocyte-specific) promoter. Suitably, the protein coding sequence is operably linked to a promoter, preferably a liver-specific (eg, hepatocyte-specific) promoter.

[0333] "Promoter" may refer to a region of DNA that causes the initiation of transcription of a gene. The promoter is located near the transcription start site of a gene, upstream of the DNA (toward the 5' region of the sense strand).

[0334] As used herein, "tissue-specific promoter" may refer to a promoter that preferentially promotes the expression of transgenic in a particular type of cell or tissue. Suitably, compared with other cell types, tissue-specific promoters can promote higher expression of transgenic in a cell type. Higher expression can be measured, for example, by measuring the expression of a transgenic (e.g., green fluorescent protein (GFP)) operably connected to a promoter, wherein the expression of the transgenic is related to the ability of the promoter to promote gene expression. For example, compared with the expression level in other cell types, a tissue-specific promoter can be a promoter that promotes the expression level of transgenic in a cell type by at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher.

[0335] In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the promoter is a liver-specific promoter.

[0336] Suitably, the promoter may be (or may be derived from) a promoter associated with a gene that has selective expression in human liver cells (e.g., hepatocytes). Suitably, the promoter may be (or may be derived from) a promoter associated with a gene that has selective expression in human liver cells. Methods for identifying promoters associated with genes will be well known to those skilled in the art.

[0337] Exemplary liver-specific and / or hepatocyte-specific promoters are described in Kattenhorn, LM, et al., 2016. Human gene therapy, 27(12), pp.947-961, and include transthyretin (TTR) promoter, alpha-1-antityrosine protease (AAT) promoter, thyroxine binding globulin (TBG) promoter, APoE / hAAT promoter, HCR-hAAT promoter, LP1 promoter, and HLP promoter.

[0338] Engineered promoter variants derived from any of these promoters may be used provided that the variant retains the ability to drive liver-specific and / or hepatocyte-specific expression of a transgene operably coupled to the promoter. The skilled artisan will arrive at such variants using methods known in the art. The variant may have at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the promoters.

[0339] Fragments of any of these promoters (or variants thereof) may be used provided that the fragment retains the ability to drive liver-specific and / or hepatocyte-specific expression of a transgene operably coupled to the promoter. The skilled artisan will be able to arrive at such fragments using methods known in the art. The length of the fragment may be, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, or at least 1000 nucleotides.

[0340] In some embodiments, the promoter is selected from the group consisting of a thyrotropin (TTR) promoter, an alpha-1-antitrypsin (AAT) promoter, a thyroxine-binding globulin (TBG) promoter, an APoE / hAAT promoter, an HCR-hAAT promoter, an LP1 promoter, and an HLP promoter.

[0341] In some embodiments, the promoter is a TTR promoter or a variant and / or fragment thereof. In some embodiments, the promoter is an Enh1mTTR (ET) promoter or a variant and / or fragment thereof.

[0342] An exemplary ET promoter is provided in GenBank Accession No. AY661265. In some embodiments, the ET promoter comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 19, or a fragment thereof. Suitably, the ET promoter comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 19, or a fragment thereof.

[0343] In some embodiments, the ET promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 19 or a fragment thereof.

[0344] CGCGAGTTAATAATTACCAGCGCGGGCCAAATAAATAATCCGCGAGGGGCAGGTGACGTTTGCCCAGCGCGCGCTGGTAATTATTAACCTCGCGAATATTGATTCGAGGCCGCGATTGCCGCAATCGCGAGGGGCAGG TGACCTTTGCCCAGCGCGCGTTCGCCCCGCCCCGGACGGTATCGATAAGCTTAGGAGCTTGGGCTGCAGGTCGAGGGCACTGGGAGGATGTTGAGTAAGATGGAAAACTACTGATGACCCTTGCAGAGACAGAGTATT AGGACATGTTTGAACAGGGGCCGGGCGATCAGCAGGTAGCTCTAGAGGATCCCCGTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTC TCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTG

[0345] Exemplary ET promoter (SEQ ID NO: 19)

[0346] In some embodiments, the promoter is an AAT promoter or a variant and / or fragment thereof. In some embodiments, the promoter is a human AAT (hAAT) promoter or a variant and / or fragment thereof.

[0347] In some embodiments, the hAAT promoter comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 20, or a fragment thereof. Suitably, the hAAT promoter comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20, or a fragment thereof.

[0348] In some embodiments, the hAAT promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 20 or a fragment thereof.

[0349] GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAG CTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAG

[0350] Exemplary hAAT promoter (SEQ ID NO: 20)

[0351] The promoter may be a constitutive promoter. As used herein, a "constitutive promoter" is a promoter that is always active.

[0352] Alternatively, the promoter can be an inducible promoter. As used herein, an "inducible promoter" is a promoter that is active only under specific conditions. For example, the expression of a transgenic can be induced by a small molecule or a drug (e.g., it binds to a promoter, a regulatory sequence, or a transcriptional repressor or activator molecule) or by using an environmental trigger. Types of inducible promoters include chemical inducible promoters (e.g., Tet-on systems); temperature inducible promoters (e.g., promoters derived from Hsp70 or Hsp90); and light inducible promoters. Suitably, the promoter is chemically inducible. Any suitable method for engineering inducible promoters can be used.

[0353] Enhancer elements

[0354] The lentiviral vector of the present invention may comprise an enhancer, preferably a liver-specific (eg, hepatocyte-specific) enhancer. Suitably, the protein coding sequence is operably linked to an enhancer, preferably a liver-specific (eg, hepatocyte-specific) enhancer.

[0355] An "enhancer" or "enhancer element" may refer to a region of DNA that can be bound by a protein (activator) to increase the likelihood that transcription of a particular gene will occur. Enhancers are cis-acting. They are located up to 1 M bp (1,000,000 bp) from a gene, either upstream or downstream of the start site.

[0356] As used herein, "tissue-specific enhancer" is an enhancer that preferentially promotes the expression of a gene in a specific cell or tissue. Suitably, compared with other cell types, tissue-specific enhancers can promote the higher expression of a gene in a specific cell type. Higher expression can be measured, for example, by measuring the expression of a transgenic (e.g., green fluorescent protein (GFP)) operably connected to an enhancer, wherein the expression of the transgenic is related to the ability of the enhancer to promote gene expression. For example, compared with the expression level in other cell types, a tissue-specific enhancer can be an enhancer that promotes the expression level of a gene in a cell type by at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher.

[0357] Suitable tissue-specific enhancers will be well known to those skilled in the art.The enhancer may be a liver-specific enhancer, preferably a hepatocyte-specific enhancer.

[0358] Suitably, the enhancer may be (or may be derived from) an enhancer associated with a gene that has selective expression in human liver cells (e.g., hepatocytes). Suitably, the enhancer may be (or may be derived from) an enhancer associated with a gene that has selective expression in human hepatocytes. Methods for identifying enhancer regions associated with genes will be well known to those skilled in the art.

[0359] Exemplary liver-specific and / or hepatocyte-specific enhancers are described in Kramer, MG, et al., 2003. Molecular therapy, 7(3), pp. 375-385, and include enhancer regions of albumin, α1-antitrypsin, hepatitis B virus core protein, and hemopexin genes. Other liver-specific and / or hepatocyte-specific enhancers include apolipoprotein E (APoE) enhancer, hepatic control region (HCR) enhancer, and α-1-antitrypsin (AAT) enhancer.

[0360] Engineered enhancer variants derived from any of these enhancers can be used, provided that the variant retains the ability to drive liver-specific and / or hepatocyte-specific expression of a transgene operably coupled to the enhancer. The skilled artisan will arrive at such variants using methods known in the art. The variant may have at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the enhancers.

[0361] Fragments of any of these enhancers (or variants thereof) may be used provided that the fragment retains the ability to drive liver-specific and / or hepatocyte-specific expression of a transgene operably coupled to the enhancer. The skilled artisan will be able to arrive at such fragments using methods known in the art. The length of the fragment may be at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, or at least 1000 nucleotides.

[0362] The vector of the present invention can comprise a liver-specific promoter and / or a liver-specific enhancer, i.e. a liver-specific promoter and / or an enhancer. Suitably, the protein coding sequence is operably linked to a liver-specific promoter and / or an enhancer. Suitably, the protein coding sequence is operably linked to a hepatocyte-specific promoter and / or an enhancer. Promoters and enhancers can be any of the above-mentioned combinations, such as hAAT promoters and ApoE or HCR enhancers.

[0363] Post-transcriptional regulatory elements

[0364] The lentiviral vector of the present invention may comprise one or more further post-transcriptional regulatory elements (e.g. in addition to one or more miRNA target sequences). Suitably, the protein coding sequence is operably linked to one or more further post-transcriptional regulatory elements. Further post-transcriptional regulatory elements may improve gene expression.

[0365] The lentiviral vector of the present invention may comprise a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Suitably, the protein coding sequence is operably linked to the WPRE.

[0366] Suitable WPRE sequences are well known to those skilled in the art (see, for example, Zufferey, R., et al., 1999. Journal of virology, 73 (4), pp. 2886-2892; and Zanta-Boussif, MA et al., 2009. Gene therapy, 16 (5), pp. 605-619). Suitably, WPRE is a wild-type WPRE or a mutant WPRE. For example, WPRE can be mutated to eliminate translation of woodchuck hepatitis virus X protein (WHX), for example by mutating the WHX ORF translation start codon.

[0367] In some embodiments, the WPRE comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 21, or a fragment thereof. Suitably, the WPRE comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 21, or a fragment thereof.

[0368] In some embodiments, the WPRE comprises or consists of the nucleotide sequence of SEQ ID NO: 21 or a fragment thereof.

[0369] AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCT TTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGGCATT GCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCC GTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGC

[0370] Exemplary WPRE (SEQ ID NO: 21)

[0371] Polyadenylation sequence

[0372] The lentiviral vector of the present invention may comprise a polyadenylation sequence. Suitably, the protein coding sequence is operably linked to the polyadenylation sequence. The polyadenylation sequence may be inserted after the protein coding sequence to improve transgene expression.

[0373] The polyadenylation sequence usually comprises a polyadenylation signal, a polyadenylation site and a downstream element: the polyadenylation signal comprises a sequence motif recognized by the RNA cleavage complex; the polyadenylation site is the site of cleavage at which the poly-A tail is added to the mRNA; the downstream element is a GT-rich region, which is usually located just downstream of the polyadenylation site and is important for efficient processing.

[0374] Suitable polyadenylation sequences will be well known to those skilled in the art (see, for example, Schambach, A., et al., 2007. Molecular Therapy, 15(6), pp. 1167-1173; and Choi, JH et al., 2014. Molecular brain, 7(1), pp. 1-10). Exemplary polyadenylation sequences include the bGH poly(A) signal sequence and the SV40pA signal sequence.

[0375] Suitably, a polyadenylation sequence may be present in the 3' LTR (ie the lentiviral vector does not comprise additional polyadenylation sequences).

[0376] Kozak sequence

[0377] The lentiviral vector of the present invention may comprise a Kozak sequence. Suitably, the protein coding sequence is operably linked to the Kozak sequence. The Kozak sequence may be inserted before the start codon to improve the initiation of translation.

[0378] Suitable Kozak sequences will be well known to those skilled in the art (see, for example, Kozak, M., 1987. Nucleic acids research, 15(20), pp. 8125-8148).

[0379] In some embodiments, the Kozak sequence comprises or consists of an amino acid sequence that is at least 80% identical to SEQ ID NO: 22, or a fragment thereof.

[0380] In some embodiments, the Kozak sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 22 or a fragment thereof.

[0381] GCCACC

[0382] Exemplary Kozak sequence (SEQ ID NO: 22)

[0383] Other cis-acting elements

[0384] The lentiviral vectors of the invention may comprise any other suitable cis-acting elements, such as one or more of the following: a rev response element (RRE); a retroviral psi packaging element; a primer binding site (PBS); a TAT activation region (TAR); splice donor and acceptor sites; and central and terminal polypurine tracts.

[0385] Long terminal repeat (LTR)

[0386] The lentiviral vector of the present invention may comprise one or more long terminal repeats (LTRs). As described above, LTRs are responsible for proviral integration and transcription. Typically, naturally occurring LTRs comprise U3, R, and U5 regions.

[0387] The lentiviral vector may comprise a 5'LTR and / or a 3'LTR. The lentiviral vector may comprise a 5'LTR and a 3'LTR. Suitably, the 5'LTR comprises the R and U5 regions, and optionally comprises the U3 region. Suitably, the 3'LTR comprises the U3, R and U5 regions.

[0388] Suitable LTR sequences will be familiar to those skilled in the art (see, eg, Frech, K., et al., 1996. Virology, 224(1), pp. 256-267).

[0389] In some embodiments, the LTR comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 23, or a fragment thereof. Suitably, the LTR comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 23, or a fragment thereof.

[0390] In some embodiments, the LTR comprises or consists of the nucleotide sequence of SEQ ID NO: 23 or a fragment thereof.

[0391] TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG

[0392] Exemplary LTR (SEQ ID NO: 23)

[0393] The lentiviral vectors of the present invention may contain one or more self-inactivating long terminal repeats (SIN-LTRs). "SIN-LTRs" may contain deletions that abolish full-length viral transcription after the full-length virus has been incorporated into the host cell. For example, the 3'SIN-LTR may contain a deletion in the U3 region that removes promoter / enhancer elements (see, e.g., Zufferey, R., et al., 1998. Journal of virology, 72 (12), pp. 9873-9880). This deletion is copied into the 5'LTR after reverse transcription, thereby making gene expression in the target cell dependent on the selected internal promoter.

[0394] Suitable SIN-LTR sequences will be well known to those skilled in the art (see, for example, Zufferey, R., et al., 1998. Journal of virology, 72(12), pp. 9873-9880 and Miyoshi, H., et al., 1998. Journal of virology, 72(10), pp. 8150-8157).

[0395] In some embodiments, the 5'LTR comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 24, or a fragment thereof. Suitably, the 5'LTR comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24, or a fragment thereof.

[0396] In some embodiments, the 5'LTR comprises or consists of the nucleotide sequence of SEQ ID NO: 24 or a fragment thereof.

[0397] GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG

[0398] Exemplary 5'LTR (SEQ ID NO:24)

[0399] In some embodiments, the 5'LTR and / or 3'LTR comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 25, or a fragment thereof. Suitably, the 5'LTR and / or 3'LTR comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 25, or a fragment thereof.

[0400] In some embodiments, the 5'LTR and / or 3'LTR comprises or consists of the nucleotide sequence of SEQ ID NO: 25 or a fragment thereof.

[0401] In some embodiments, the 5'LTR and 3'LTR comprise or consist of an amino acid sequence at least 70% identical to SEQ ID NO: 25, or a fragment thereof. Suitably, the 5'LTR and 3'LTR comprise or consist of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 25, or a fragment thereof.

[0402] In some embodiments, the 5'LTR and the 3'LTR comprise or consist of the nucleotide sequence of SEQ ID NO: 25 or a fragment thereof.

[0403] TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG

[0404] Exemplary 3'LTR (SEQ ID NO:25)

[0405] Primer Binding Site (PBS)

[0406] The lentiviral vector of the present invention may contain a primer binding site (PBS). The PBS is a cis-acting element to which a primer can bind to initiate reverse transcription of the RNA genome (see, for example, Lanchy, JM, et al., 1998. Journal of Biological Chemistry, 273 (38), pp. 24425-24432).

[0407] Suitable retroviral PBSs will be well known to those skilled in the art.

[0408] In some embodiments, the PBS comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 26, or a fragment thereof. Suitably, the PBS comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 26, or a fragment thereof.

[0409] In some embodiments, the PBS comprises or consists of the nucleotide sequence SEQ ID NO: 26 or a fragment thereof.

[0410] TGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAG

[0411] Exemplary primer binding site (SEQ ID NO: 26)

[0412] In some embodiments, the PBS comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 27, or a fragment thereof. Suitably, the PBS comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 27, or a fragment thereof.

[0413] In some embodiments, the PBS comprises or consists of the nucleotide sequence SEQ ID NO: 27 or a fragment thereof.

[0414] TGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAG

[0415] Exemplary primer binding site (SEQ ID NO: 27)

[0416] Retroviral psi packaging element

[0417] The lentiviral vector of the present invention may comprise a retroviral psi packaging element. As described above, the retroviral psi packaging element is a cis-acting element that is involved in regulating the essential process of packaging the retroviral RNA genome into the viral capsid during replication (see, e.g., McBride, MS, et al., 1997. Journal of virology, 71(6), pp. 4544-4554). The retroviral psi packaging element may form part of the 5' region of the gag gene.

[0418] Suitable retroviral psi packaging elements will be well known to those skilled in the art.

[0419] In some embodiments, the retroviral psi packaging element comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 28, or a fragment thereof. Suitably, the retroviral psi packaging element comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 28, or a fragment thereof.

[0420] In some embodiments, the retroviral psi packaging element comprises or consists of the nucleotide sequence of SEQ ID NO: 28 or a fragment thereof.

[0421] ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATA CTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGAT

[0422] Exemplary retroviral psi packaging element (SEQ ID NO: 28)

[0423] Rev response element (RRE)

[0424] The lentiviral vector of the present invention may contain a rev response element (RRE). As described above, RRE is a cis-acting element that enables the RNA transcripts of the integrated provirus to be efficiently exported from the nucleus of the infected target cell to the cytoplasm (see, for example, Pollard, VW and Malim, MH, 1998. Annual review of microbiology, 52 (1), pp. 491-532).

[0425] Suitable RRE sequences will be well known to those skilled in the art.

[0426] In some embodiments, the RRE comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 29, or a fragment thereof. Suitably, the RRE comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 29, or a fragment thereof.

[0427] In some embodiments, the RRE comprises or consists of the nucleotide sequence of SEQ ID NO: 29 or a fragment thereof.

[0428] GGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGG CTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTT Exemplary rev response element (SEQ ID NO:29)

[0429] In some embodiments, the RRE comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 30, or a fragment thereof. Suitably, the RRE comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 30, or a fragment thereof.

[0430] In some embodiments, the RRE comprises or consists of the nucleotide sequence of SEQ ID NO: 30 or a fragment thereof.

[0431] GGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGG CTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTT Exemplary rev response element (SEQ ID NO:30)

[0432] Central polypurinergic tract (cPPT)

[0433] The lentiviral vector of the present invention may comprise a central polypurine tract (cPPT). As described above, the cPPT may allow initiation of plus strand synthesis (see, for example, Follenzi, A. et al., 2000. Nature genetics, 25(2), pp. 217-222).

[0434] Suitable cPPT sequences are well known to those skilled in the art.

[0435] In some embodiments, the cPPT comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 31, or a fragment thereof. Suitably, the cPPT comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 31, or a fragment thereof.

[0436] In some embodiments, the cPPT comprises or consists of the nucleotide sequence SEQ ID NO: 31 or a fragment thereof.

[0437] AACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATC

[0438] Exemplary central polypurine tract (SEQ ID NO: 31)

[0439] Other components

[0440] The lentiviral vector of the present invention may comprise any other suitable elements.

[0441] In some embodiments, the lentiviral vector of the present invention comprises an element comprising or consisting of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 32, or a fragment thereof. In some embodiments, the lentiviral vector of the present invention comprises an element comprising or consisting of a nucleotide sequence of SEQ ID NO: 32, or a fragment thereof.

[0442] TCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATA

[0443] Exemplary deltaENV1 (SEQ ID NO: 32)

[0444] In some embodiments, the lentiviral vector of the present invention comprises an element comprising or consisting of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 33, or a fragment thereof. In some embodiments, the lentiviral vector of the present invention comprises an element comprising or consisting of a nucleotide sequence of SEQ ID NO: 33, or a fragment thereof.

[0445] GGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGCAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAA CATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC

[0446] Exemplary deltaENV2 (SEQ ID NO: 33)

[0447] Exemplary cis-acting elements

[0448] The lentiviral vector of the present invention may comprise a cis-acting element comprising a PBS, a retroviral psi packaging element and a rev response element (RRE).

[0449] In some embodiments, the cis-acting elements comprising the PBS, the retroviral psi packaging elements, and the RRE comprise or consist of a nucleotide sequence at least 70% identical to SEQ ID NO: 34, or a fragment thereof. Suitably, the cis-acting elements comprising the PBS, the retroviral psi packaging elements, and the RRE comprise or consist of a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 34, or a fragment thereof.

[0450] In some embodiments, the cis-acting elements comprising the PBS, the retroviral psi packaging element, and the RRE comprise or consist of the nucleotide sequence of SEQ ID NO: 34 or a fragment thereof.

[0451] TGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTT

[0452] Exemplary cis - acting elements containing PBS, retroviral psi packaging element and RRE (SEQ ID NO: 34)

[0453] The lentiviral vector of the present invention may comprise a cis-acting element comprising a PBS, a retroviral psi packaging element, a rev response element (RRE), and a central polypurine tract (cPPT).

[0454] In some embodiments, the cis-acting elements comprising the PBS, the retroviral psi packaging elements, the RRE and the cPPT comprise or consist of a nucleotide sequence at least 70% identical to SEQ ID NO: 35, or a fragment thereof. Suitably, the cis-acting elements comprising the PBS, the retroviral psi packaging elements, the RRE and the cPPT comprise or consist of a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 35, or a fragment thereof.

[0455] In some embodiments, the cis-acting elements comprising the PBS, the retroviral psi packaging element, the RRE, and the cPPT comprise or consist of the nucleotide sequence of SEQ ID NO: 35 or a fragment thereof.

[0456]

[0457] Exemplary cis-acting elements comprising PBS, retroviral psi packaging element, RRE and cPPT (SEQ ID NO: 35)

[0458] Exemplary Lentiviral Genomes

[0459] The lentiviral genome of the present invention may comprise, from 5' to 3', a 5' LTR, one or more cis-acting elements, a protein coding sequence, optionally one or more post-transcriptional regulatory sequences, and a 3' LTR.

[0460] For example, the lentiviral genome of the present invention may comprise, from 5' to 3', 5' LTR, PBS, retroviral psi packaging element, RRE, cPPT, liver-specific promoter, protein coding sequence, WPRE, one or more miRNA target sequences and 3' LTR.

[0461] The lentiviral genome of the present invention may further comprise any other suitable elements, such as any other elements described herein or one or more spacer sequences. The spacer sequence may comprise, for example, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten nucleotide bases.

[0462] In some embodiments, the lentiviral genome comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 36, or a fragment thereof. Suitably, the lentiviral genome comprises or consists of a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 36, or a fragment thereof.

[0463] In some embodiments, the lentiviral genome comprises or consists of the nucleotide sequence of SEQ ID NO: 36 or a fragment thereof.

[0464]

[0465] Exemplary lentiviral genome (SEQ ID NO: 36)

[0466] Variants, derivatives, analogs and fragments

[0467] In addition to the specific polypeptides and polynucleotides mentioned herein, the present invention also encompasses variants, derivatives and fragments thereof.

[0468] In the context of the present invention, a "variant" of any given sequence refers to a sequence in which a particular sequence of residues (whether amino acids or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question retains at least one or all of its endogenous functions. Variant sequences may be obtained by addition, deletion, substitution, modification, replacement and / or alteration of at least one residue present in a naturally occurring polypeptide or polynucleotide.

[0469] As used herein, the term "derivative" in relation to a protein or polypeptide of the invention includes any substitution, variation, modification, replacement, deletion and / or addition from or to one (or more) amino acid residues in the sequence, provided that the resulting protein or polypeptide retains at least one or all of its endogenous functions.

[0470] Generally, amino acid substitutions (eg, from 1, 2 or 3 to 10 or 20 substitutions) can be made, provided that the modified sequence retains the desired activity or ability.Amino acid substitutions may include the use of non-naturally occurring analogs.

[0471] The polypeptides used in the present invention may also have deletions, insertions or substitutions of amino acid residues, which produce silent changes and result in functionally equivalent polypeptides. Deliberate amino acid substitutions may be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues, as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups of similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0472] Conservative substitutions can be made, for example according to the following table. Amino acids in the same block in the second column and amino acids in the same row in the third column can be substituted for each other:

[0473]

[0474] The effects of addition, deletion, substitution, modification, replacement and / or variation can be predicted using any suitable prediction tool such as SIFT (Vaser, R., et al., 2016. Nature protocols, 11 (1), pp. 1-9), PolyPhen-2 (Adzhubei, I., et al., 2013. Current protocols in human genetics, 76 (1), pp. 7-20), CADD (Rentzsch, P., et al., 2021. Genome medicine, 13 (1), pp. 1-12), REVEL (Ioannidis, NM, et al., 2016. The American Journal of Human Genetics, 99 (4), pp. 877-885), MetaLR (Dong, C., et al., 2015. Human molecular genetics, 24 (8), pp. 2125-2137) and / or MutationAssessor (Reva, B., et al., 2011. Nucleic acids Research, 39 (17), pp. e118-e118) or predicted based on clinical data such as ClinVar (Landrum, MJ, et al., 2016. Nucleic acids research, 44 (D1), pp. D862-D868). Suitable additions, deletions, substitutions, modifications, replacements and / or variations can be considered tolerable, benign and / or likely to be benign.

[0475] Generally, a variant may have a certain identity with a wild-type amino acid sequence or a wild-type nucleotide sequence.

[0476] In the present context, variant sequences are considered to include amino acid sequences that may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although variants may also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferred to express in terms of sequence identity.

[0477] In this context, variant sequences are considered to include nucleotide sequences that may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although variants may also be considered with respect to similarity, in the context of the present invention, it is preferred to express variants with respect to sequence identity.

[0478] Suitably, reference to a sequence having a percentage identity to any one of the SEQ ID NOs detailed herein refers to the sequence having that percentage identity over the entire length of the referenced SEQ ID NO.

[0479] Sequence identity comparisons can be performed by eye, or more usually, with the aid of available sequence comparison programs. These commercially available computer programs can calculate the percent identity between two or more sequences.

[0480] Percent identity can be calculated over contiguous sequences by aligning one sequence with the other and directly comparing each amino acid or nucleotide in one sequence with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called a "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0481] While this is a very simple and consistent approach, it does not take into account that, for example, in a pair of otherwise identical sequences, an insertion or deletion in an amino acid or nucleotide sequence can result in the subsequent residue or codon being placed out of the alignment, thus potentially resulting in a substantial reduction in percent identity when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions at the expense of overly penalizing the overall identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local identity.

[0482] In some embodiments, the present invention relates to the comparison of the sequence alignment of amino acid sequence with the same number of gaps. However, these more complex methods assign "gap penalties" to each gap present in the comparison, so that for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible (reflecting the higher correlation between the two compared sequences) will achieve a higher score than a sequence alignment with many gaps. Usually an "affine gap cost" is used, which charges a relatively high cost to the existence of a gap, and charges a smaller penalty to each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will produce an optimized comparison with less gaps. Most alignment programs allow modification of gap penalties. However, when using this type of software to compare sequences, it is preferred to use default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is gap-12 and each extension-4.

[0483] Therefore, calculation of the maximum percent identity first requires producing an optimal alignment, which takes into account the gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (see, for example, Devereux, J., et al., 1984. Nucleic acids research, 12(1), pp. 387-395). Examples of other software that can perform sequence comparisons include, but are not limited to, BLAST packages (see, e.g., Altschul, SF, et al., 1990. Journal of molecular biology, 215 (3), pp. 403-410), BLAST 2 (see, e.g., Tatusova, TA and Madden, TL, 1999. FEMS microbiology letters, 174 (2), pp. 247-250), FASTA (see, e.g., Pearson, WR and Lipman, DJ, 1988. PNAS, 85 (8), pp. 2444-2448.), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47 (W1), pp. W636-W641) and the GENEWORKS alignment tool suite. For some applications, it is preferred to use EMBOSS Needle.

[0484] Although the final percent identity can be measured, the alignment process itself is usually not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​often used, assigning scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix.

[0485] Once the software has produced the optimal alignment, it is possible to calculate the percentage of sequence identity. The software typically performs this as part of a sequence comparison and generates a numerical result. The percentage of sequence identity can be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0486] "Fragment" is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or in, for example, an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0487] Such variants, derivatives and fragments can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertion is required, synthetic DNA encoding the insert can be prepared, together with 5' and 3' flanking regions corresponding to the naturally occurring sequences on both sides of the insertion site. The flanking regions will contain convenient restriction sites corresponding to the sites in the naturally occurring sequence, so that the sequence can be cut with an appropriate enzyme and the synthetic DNA can be connected to the nick. The DNA is then expressed according to the present invention to prepare the encoded polypeptide. These methods only illustrate many standard techniques known in the art for manipulating DNA sequences, as well as other known techniques that can be used.

[0488] Generation method

[0489] In one aspect, the invention provides a method of producing a lentiviral vector according to the invention.

[0490] Suitable methods for producing lentiviral vectors will be well known to those skilled in the art (see, for example, Merten, OW, et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p. 16017).

[0491] The production method may comprise: (a) introducing a transfer vector and one or more helper vectors into a host cell; (b) culturing the host cell under conditions suitable for producing a lentiviral vector according to the present invention; and (c) obtaining the lentiviral vector from the host cell.

[0492] As used herein, a "transfer vector" may encode a lentiviral genome of the invention. Suitably, a transfer vector for producing a lentiviral genome in a host cell / packaging cell will have sufficient lentiviral genetic information to allow the RNA genome to be packaged into viral particles in the presence of packaging components (e.g., gag-pol, rev, env) that are capable of infecting target cells but cannot replicate independently to produce infectious viral particles in the final target cell.

[0493] The transfer vector for producing the viral genome in the host cell / packaging cell may include a transcriptional regulatory control sequence operably linked to the lentiviral genome to direct the genome to be transcribed in the host cell / packaging cell. These regulatory sequences may be natural sequences associated with the transcribed viral sequence (i.e., 5'U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g., CMV promoter). The transfer vector may be a plasmid.

[0494] As used herein, "auxiliary vector" can encode one or more packaging components (e.g., gag-pol, rev, env). The nucleotide sequence encoding the packaging components can be operably linked to a promoter (e.g., a CMV promoter or an RSV promoter) and / or a polyadenylation signal. The term "auxiliary vector" can include a "packaging vector" (e.g., encoding gag-pol or rev) and an "envelope vector" (e.g., encoding an env gene, such as VSV-g). The auxiliary vector, packaging vector, and / or envelope vector can be a plasmid.

[0495] The transfer vector and one or more auxiliary vectors can be introduced into the host cell by any suitable technique known in the art such as transfection, transduction and / or transformation. Suitably, the auxiliary vector can be transiently transfected or transduced into the host cell, or can be stably maintained in the host cell (e.g., stably integrated into the cell genome or maintained as an episome (episomally)). Alternatively, the auxiliary vector can be introduced into the host cell using a combination of transient transfection or transduction and stable maintenance.

[0496] Suitably, the transfer vector and / or the auxiliary vector can be a plasmid and introduced by transfection. Suitably, a four-plasmid system consisting of a transfer plasmid and three auxiliary plasmids can be used. The three auxiliary plasmids can be composed of: a first auxiliary plasmid encoding a gag-pol gene; a second auxiliary plasmid encoding a rev gene; and a third auxiliary plasmid encoding an env gene. Alternatively, a three-plasmid system can be used, which consists of a transfer plasmid, an auxiliary plasmid encoding a gag-pol gene and a rev gene; and an auxiliary plasmid encoding an env gene. Alternatively, a two-plasmid system can be used, in which all auxiliary functions (e.g., gag-pol, rev, and env) are encoded by an auxiliary plasmid.

[0497] Any suitable host cell can be used to produce a lentiviral vector. Suitable host cells include production cells and packaging cells, such as those described below (e.g., HEK 293 or derivatives thereof). Suitable conditions for culturing host cells will be well known to the technician. For example, host cells can be incubated in a chemically defined culture medium for about 1 day to about 5 days (e.g., about 48 hours, about 72 hours, or about 96 hours).

[0498] The lentiviral vector can be obtained using any suitable method known in the art. For example, the culture supernatant can be harvested, and the lentiviral vector can then be purified from the culture supernatant (e.g., by centrifugation, membrane filtration and / or chromatography). The production method can further include any other suitable process steps, such as DNA reduction, concentration, formulation and / or sterilization.

[0499] Vectors, kits and systems

[0500] In one aspect, the invention provides a vector encoding a lentiviral genome of the invention. The vector may be a transfer vector as described herein. For example, the vector may be a plasmid and / or the lentiviral genome may be operably linked to a promoter (e.g., a viral promoter, such as a CMV promoter).

[0501] In one aspect, the present invention provides a kit or system for producing a lentiviral vector of the present invention.

[0502] The kit or system may be a lentiviral packaging kit or system or a lentiviral production kit or system. As used herein, a "lentiviral packaging kit or system" may include one or more components for packaging a lentiviral vector of the invention, and optionally instructions. As used herein, a "lentiviral production kit or system" may include one or more components for producing a lentiviral vector of the invention, and optionally instructions.

[0503] The kit or system may include a transfer vector encoding the lentiviral genome of the present invention, and optionally one or more auxiliary vectors. The kit or system may further include host cells (e.g., packaging cells or production cells) and / or other reagents (e.g., transfection reagents, culture media, etc.). The kit or system may further include any other suitable components for packaging and / or producing the lentiviral vector of the present invention, and optionally instructions.

[0504] cell

[0505] In one aspect, the invention provides a cell comprising a lentiviral vector of the invention. The cell may be an isolated cell. Suitably, the cell is a mammalian cell, such as a human cell. The cell may be an isolated human cell.

[0506] Suitably, the cell can be a production cell. The term "production cell" is included in the cells producing viral particles after transient transfection, stable transfection or vector transduction of all elements necessary for producing viral particles, or any cell engineered to stably contain the elements necessary for producing viral particles. Suitable production cells are well known to those skilled in the art, and can include HEK293, COS-1, COS-7, CV-1, HeLa, CHO and A549 cell lines. In some embodiments, the production cell is HEK293 cell or its derivative (e.g., HEK293T cell, HEK293T Lenti-X, HEK293T-Rex cell, HEK293FT cell, HEK293SF-3F6 cell, HEK293SF-3F9 cell, HEK293-EBNA1 cell or SJ293TS cell).

[0507] Suitably, the cell can be a packaging cell. The term "packaging cell" includes cells containing some or all of the elements necessary for packaging recombinant viral genomes. Typically, such packaging cells contain one or more vectors capable of expressing viral structural proteins (e.g., gag-pol, rev, env) and / or one or more genes encoding viral structural proteins have been integrated into the genome of the packaging cell. After subsequent steps of transient transfection, transduction or stable integration of each additional required element, cells containing only some of the elements required for producing enveloped viral particles can be used as intermediates for generating viral particle production cell lines. The term "packaging cell" encompasses these intermediates. Suitable packaging cells will be well known to those skilled in the art (see, e.g., Merten, OW, et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p. 16017).

[0508] Suitably, the cell can be a liver cell, such as a hepatocyte. Suitably, the cell can be an immortalized liver cell, such as an immortalized hepatocyte. Suitable cell lines will be well known to those skilled in the art, such as HepG2, Hep3B, HBG and HepaRG cell lines. Methods for generating immortalized liver cells (such as immortalized hepatocytes) are well known to those skilled in the art (see, for example, Ramboer, E., et al., 2015. Methods Mol Biol, 1250, pp. 53-76). Suitably, the cell can be a stem cell.

[0509] Pharmaceutical composition

[0510] In one aspect, the invention provides a pharmaceutical composition comprising the lentiviral vector or cell of the invention. In a preferred embodiment, the pharmaceutical composition comprises the lentiviral vector of the invention in the form of a lentiviral particle.

[0511] A pharmaceutical composition is a composition comprising or consisting of a therapeutically effective amount of a pharmaceutically active agent (eg, a lentiviral vector). The pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).

[0512] By "pharmaceutically acceptable" is meant that the formulation is sterile and pyrogen-free. The carrier, diluent and / or excipient must be "acceptable" in the sense of being compatible with the lentiviral vector and not harmful to the recipient thereof. Typically, the carrier, diluent and excipient will be saline or an infusion medium that is sterile and pyrogen-free, however, other acceptable carriers, diluents and excipients may be used.

[0513] Acceptable carriers, diluents and excipients for therapeutic use are well known in the pharmaceutical field. The choice of pharmaceutical carrier, excipient or diluent can be selected with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable binder, lubricant, suspending agent, coating agent or solubilizing agent as a carrier, excipient or diluent (or in addition to a carrier, excipient or diluent).

[0514] Examples of pharmaceutically acceptable carriers include, for example, water, saline solutions, alcohol, silicone, wax, vaseline, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oils, fatty acid monoglycerides and diglycerides, petroleum ether fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0515] The lentiviral vector, cell or pharmaceutical composition according to the present invention can be administered in a manner suitable for treating and / or preventing the diseases described herein. Suitable routes of administration are known to those skilled in the art (see, for example, Fumoto, S., et al., 2013. Novel Gene Therapy Approaches, pp. 3-31). The amount and frequency of administration can be determined by a technician, for example, depending on factors such as the condition of the subject and the type and severity of the subject's disease. The pharmaceutical composition can be formulated accordingly.

[0516] The lentiviral vector, cell or pharmaceutical composition according to the present invention can be administered parenterally (e.g., intravenously, intra-arterially, intramuscularly, intrathecally, subcutaneously) or by infusion techniques. The lentiviral vector, cell or pharmaceutical composition can be administered in the form of a sterile aqueous solution, which may contain other substances, such as sufficient salt or glucose to make the solution isotonic with the blood. The aqueous solution can be appropriately buffered (preferably to a pH of 3 to 9). The pharmaceutical composition can be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions is easily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0517] The lentiviral vector, cell or pharmaceutical composition according to the present invention can be administered systemically, for example by intravenous injection or intraperitoneal injection. In some embodiments, the lentiviral vector, cell or pharmaceutical composition according to the present invention is administered by intravenous injection. The pharmaceutical composition can be formulated accordingly.

[0518] The lentiviral vector, cell or pharmaceutical composition according to the present invention can be administered locally, for example by direct injection, intra-arterial injection or intra-portal injection. In some embodiments, the lentiviral vector, cell or pharmaceutical composition according to the present invention is administered locally to the liver. In some embodiments, the lentiviral vector, cell or pharmaceutical composition according to the present invention is administered by intrahepatic injection, intrahepatic arterial injection or intra-portal injection. The pharmaceutical composition can be formulated accordingly.

[0519] The pharmaceutical composition may comprise the lentiviral vector or cell of the invention in an infusion medium (eg, a sterile isotonic solution). The pharmaceutical composition may be packaged in an ampoule, a disposable syringe or a multiple-dose vial made of glass or plastic.

[0520] The lentiviral vector, cell or pharmaceutical composition can be administered in a single dose or multiple doses. Suitably, the lentiviral vector, cell or pharmaceutical composition can be administered in a single, one-time dose. The pharmaceutical composition can be formulated accordingly.

[0521] The lentiviral vector, cell or pharmaceutical composition can be administered at different doses (e.g., measured in transducing units (TU) / kg). In any case, the physician can determine the actual dose that will be most suitable for any individual subject, and the dose may vary, for example, with the age, weight and response of the particular subject. Suitably, the lentiviral vector of the invention is administered at a dose of at least about 10 8 TU / kg, at least about 10 9 TU / kg, or at least about 10 10 Suitably, the lentiviral vector of the present invention is administered at a dose of about 10 TU / kg. 13 TU / kg or less, about 10 12 TU / kg or less, or about 1011 TU / kg or less. Suitably, the lentiviral vector of the present invention is administered at a dose of about 10 8 to about 10 13 TU / kg, about 10 9 to about 10 13 TU / kg or about 10 10 to about 10 13 Suitably, the lentiviral vector of the present invention is administered at a dose of about 10 TU / kg. 8 to about 10 12 TU / kg, about 10 9 to about 10 12 TU / kg or about 10 10 to about 10 12 Suitably, the lentiviral vector of the present invention is administered at a dose of about 10 TU / kg. 8 to about 10 11 TU / kg, about 10 9 to about 10 11 TU / kg or about 10 10 to about 10 11 In some embodiments, the lentiviral vector of the present invention is administered at a dose of about 10 TU / kg. 8 to about 10 11 TU / kg, about 10 8 to about 10 10 TU / kg, or about 10 9 to about 10 10 In some embodiments, the lentiviral vector of the present invention is administered at a dose of about 10 TU / kg. 9 to about 10 10 The pharmaceutical composition can be formulated accordingly.

[0522] The pharmaceutical composition may further comprise one or more other therapeutic agents.

[0523] The invention further includes a kit comprising the lentiviral vector, cell and / or pharmaceutical composition of the invention. Preferably, the kit is used in the method as described herein and is used as described herein, e.g., a therapeutic method as described herein. Preferably, the kit comprises instructions for using the kit components.

[0524] Methods for treating and / or preventing disease

[0525] In one aspect, the invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the invention for use as a medicament.

[0526] In one aspect, the present invention provides use of the lentiviral vector, cell or pharmaceutical composition according to the present invention in the preparation of a medicament.

[0527] In one aspect, the present invention provides a method of administering a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0528] The lentiviral vector-mediated gene therapy described herein can allow stable gene transfer in pediatric patients even in the first disease stages by virtue of lentiviral vector genome integration.

[0529] After administering the lentiviral vector of the present invention, the lentiviral vector can be integrated into the genome of liver cells (e.g., hepatocytes). Subsequently, the lentiviral vector can be maintained in the genome of liver cells (e.g., hepatocytes) when they replicate. The integration of the lentiviral vector in the liver cell genome can be determined by integration site (IS) analysis (e.g., quantitative high-throughput vector IS analysis). Suitable methods are known in the art (see, e.g., Cantore, A., et al., 2015. Science translational medicine, 7 (277), p. 277ra28).

[0530] The lentiviral vector, cell or pharmaceutical composition can be administered to any subject in need thereof. The subject can be a mammal (e.g., a human). In some embodiments, the subject is male. In some embodiments, the subject is female. In a preferred embodiment, the lentiviral vector is administered in the form of lentiviral particles.

[0531] In some embodiments, the subject is a juvenile, an adolescent, or a child. The term "juvenile" may refer to an individual who has not yet reached adulthood. The term "adolescent" may refer to an individual during the period from the beginning of puberty to adulthood. The term "child" may refer to an individual between the birth and puberty stages.

[0532] In some embodiments, the subject is a young child, a toddler, an infant. The term "young child" may refer to a human subject aged from 3 to 5 years old. The term "toddler" may refer to a human subject aged from 1 to 3 years old. The term "infant" may be intended to refer to a human subject under 12 months old.

[0533] In some embodiments, the subject is a pediatric patient. The term "pediatric patient" can refer to a human subject up to about 18-21 years of age (see, e.g., Sawyer, SM, et al., 2019. The Lancet Child & Adolescent Health, 3(11), pp. 822-830).

[0534] In some embodiments, the subject is a neonatal patient or an infant patient. The term "neonatal patient" may refer to a human subject whose age is about 4 weeks old or younger. The term "infant patient" may refer to a human subject whose age is about 4 weeks to about 1 year old.

[0535] In other embodiments, the subject is an adult.In humans, the human liver is expected to completely renew itself every 5 years, so integrative vectors are expected to be more durable than most episomal vectors (eg, AAV).

[0536] The lentiviral vector-mediated gene therapy described herein can allow long-term treatment and / or prevention (e.g., after a single, one-time dose administration). For example, the vector-mediated gene therapy described herein can be effective for a period of at least about 6 months or more, at least about 7 months or more, at least about 8 months or more, at least about 9 months or more, at least about 10 months or more, at least about 11 months or more, or at least about 1 year or more.

[0537] Methylmalonic acidemia (MMA)

[0538] The vector, cell or pharmaceutical composition according to the present invention can be used to prevent and / or treat methylmalonic acidemia (MMA).

[0539] In one aspect, the present invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the present invention for use in preventing or treating methylmalonic acidemia (MMA).

[0540] In one aspect, the present invention provides use of a lentiviral vector, a cell or a pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating methylmalonic acidemia (MMA).

[0541] In one aspect, the present invention provides a method for preventing or treating methylmalonic acidemia (MMA), comprising administering a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0542] As mentioned above, methylmalonic acidemia (MMA), also known as methylmalonic aciduria, is a group of inborn errors of metabolism associated with elevated concentrations of methylmalonic acid in the blood and urine, which results from the failure to convert methylmalonyl-CoA to succinyl-CoA during propionyl-CoA metabolism in the mitochondrial matrix.

[0543] In preferred embodiments, MMA is isolated MMA (iMMA). As used herein, "isolated MMA" or "iMMA" may refer to MMA without hyperhomocysteinemia or homocystinuria, hypomethioninemia or other metabolites such as changes in malonate (see, e.g., Manoli I, et al. 2005. Isolated Methylmalonic Acidemia. In Gene Reviews).

[0544] MMA and iMMA can be divided into subgroups based on genetic defects, clinical presentation, and / or laboratory findings (see e.g. F., et al., 2007. Pediatric research, 62(2), pp.225-230; and Keyfi, F., et al., 2016. Reports of biochemistry & molecular biology, 5(1), pp.1-14). Subtypes may include mut-MMA (mut-MMA), cblA-MMA (cblA-MMA), cblB-MMA (cblB-MMA), cblD-MMA (cblD-MMA) and MCEE deficiency.

[0545] Typically, the mut form of MMA is unresponsive to vitamin B12 therapy. Patients with defects in AdoCbl synthesis are usually responsive to vitamin B12 therapy and are classified as 'cbl' type. A definitive diagnosis of MMA can be based on urine organic acid analysis using gas chromatography / mass spectrometry. Patients with mut-MMA may have a urine methylmalonic acid concentration of about 1000 to about 10000 mmol / mol creatinine; patients with cbl-MMA may have a urine methylmalonic acid concentration of about 10 to about 100 mmol / mol creatinine; and / or patients with MCEE deficiency may have a urine methylmalonic acid concentration of about 50 to about 1500 mmol / mol creatinine. MMA subtypes can be further diagnosed by enzyme assay analysis and / or molecular studies (see, e.g., Keyfi, F., et al., 2016. Reports of biochemistry & molecular biology, 5 (1), pp. 1-14).

[0546] In some embodiments, the MMA is selected from the group consisting of mut-MMA, cblA-MMA, cblB-MMA, cblD-MMA, and MCEE deficiency. In preferred embodiments, the MMA is mut-MMA.

[0547] After administering the lentiviral vector of the present invention to a subject in need thereof, the plasma methylmalonic acid level can be prevented from increasing or decreasing. Suitably, the plasma methylmalonic acid level can be reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. Suitably, after administration, the plasma methylmalonic acid level of the subject can be less than about 500 μmol / L, or less than about 200 μmol / L. After administering the lentiviral vector of the present invention to a subject in need thereof, the plasma methylmalonic acid level can be normalized. Normal plasma methylmalonic acid levels can be <0.27 μmol / L (see, e.g., Manoli I, et al. 2005. Isolated Methylmalonic Acidemia. In Gene Reviews). Plasma methylmalonic acid levels can be determined by any method known in the art (e.g., as described in Rizzo, C., et al., 2014. Clinica chimica acta, 429, pp. 30-33).

[0548] After administering the lentiviral vector of the present invention to a subject in need thereof, the urine methylmalonic acid level can be prevented from increasing or decreasing. Suitably, the urine methylmalonic acid level can be reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. Suitably, after administration, the urine methylmalonic acid level of the subject can be less than about 5000mmol / molCr, less than about 2000mmol / molCr or less than about 1000mmol / molCr. After administering the lentiviral vector of the present invention to a subject in need thereof, the urine methylmalonic acid level can be normalized. Normal urine methylmalonic acid levels can be <4mmol / molC (see, e.g., Manoli I, et al. 2005.Isolated Methylmalonic Acidemia.InGeneReviews). Urine methylmalonic acid levels can be determined by any method known in the art (e.g., as described in Rasmussen, K., 1989.Clinical chemistry, 35 (2), pp.260-264).

[0549] After administering the lentiviral vector of the present invention to a subject in need thereof, the level of methylmalonic acid in the liver can be prevented from increasing or decreasing. Suitably, the level of methylmalonic acid in the liver can be reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. Suitably, after administration, the level of methylmalonic acid in the liver of the subject can be less than about 50 μmol / L, less than about 10 μmol / L or less than about 5 μmol / L. After administering the lentiviral vector of the present invention to a subject in need thereof, the level of methylmalonic acid in the liver can be normalized. The level of methylmalonic acid in the liver can be determined by any method known in the art (e.g., as disclosed herein).

[0550] After administering the lentiviral vector of the present invention to a subject in need thereof, the kidney methylmalonic acid level can be prevented from increasing or decreasing. Suitably, the kidney methylmalonic acid level can be reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. Suitably, after administration, the subject's kidney methylmalonic acid level can be less than about 100 μmol / L, or less than about 50 μmol / L. After administering the lentiviral vector of the present invention to a subject in need thereof, the kidney methylmalonic acid level can be normalized. The kidney methylmalonic acid level can be determined by any method known in the art (e.g., as disclosed herein).

[0551] After administering the lentiviral vector of the present invention to a subject in need thereof, brain methylmalonic acid levels can be prevented from increasing or decreasing. Suitably, brain methylmalonic acid levels can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. Suitably, after administration, the subject's brain methylmalonic acid level can be less than about 25 μmol / L, or less than about 20 μmol / L. After administering the lentiviral vector of the present invention to a subject in need thereof, brain methylmalonic acid levels can be normalized. Brain methylmalonic acid levels can be determined by any method known in the art (e.g., as disclosed herein).

[0552] After applying the lentiviral vector of the present invention to a subject in need, the mitochondrial function of the subject can be improved.Mitochondrial function can be determined by any suitable method known in the art, for example, by measuring biochemical markers such as mitochondrial membrane potential, lactate / ester, pyruvate / ester, creatine kinase, acylcarnitine and amino acids (e.g., alanine, glycine, proline and threonine), see, for example, Hubens, WHG, et al., 2021.Mitochondrion, 62, pp.187-204. Suitably, after applying the lentiviral vector of the present invention to a subject in need, plasma FGF21 levels can be prevented from increasing or decreasing. Suitably, plasma FGF21 levels can be reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. Suitably, after application, the plasma FGF21 level of the subject can be less than about 1000pg / ml. After applying the lentiviral vector of the present invention to a subject in need, plasma FGF21 levels can be normalized. Normal plasma FGF21 levels may be <200 pg / ml. Plasma FGF21 levels may be determined by any method known in the art, such as described in Manoli, I., et al., 2018. JCI insight, 3(23), e124351.

[0553] After administering the lentiviral vector of the present invention to a subject in need thereof, hepatic mitochondrial disease, renal mitochondrial disease and / or hepatorenal mitochondrial disease can be improved. After administering the lentiviral vector of the present invention to a subject in need thereof, the formation of giant mitochondria (megamitochondria) can be reduced and / or prevented (e.g., in the liver). The progression of hepatic mitochondrial disease, renal mitochondrial disease and / or hepatorenal mitochondrial disease and / or the formation of giant mitochondria can be determined by any suitable method known in the art (see, e.g., Chandler, RJ, et al., 2009. The FASEB Journal, 23 (4), pp. 1252-1261), e.g., by electron microscopy analysis. Suitably, after administering the lentiviral vector of the present invention to a subject in need thereof, the mitochondrial area, circumference and / or shape of mitochondria in the liver are normalized (e.g., any difference compared to a phenotypically normal subject may be statistically insignificant). Suitably, following administration of a lentiviral vector of the invention to a subject in need thereof, the number of circular structures in mitochondria in the kidney is normalized (eg, any difference compared to a phenotypically normal subject may be statistically insignificant).

[0554] After administering the lentiviral vector of the present invention to a subject in need thereof, glutathione deficiency (e.g., in the liver) can be reduced and / or prevented. After administering the lentiviral vector of the present invention to a subject in need thereof, glutathione levels (e.g., liver glutathione levels) can be normalized. Glutathione levels can be determined by any suitable method known in the art (see, e.g., Chandler, RJ, et al., 2009. The FASEB Journal, 23(4), pp. 1252-1261).

[0555] After administering the lentiviral vector of the present invention to a subject in need thereof, undesirable growth can be reduced and / or prevented. After administering the lentiviral vector of the present invention to a subject in need thereof, growth can be normalized. Growth can be determined by any suitable method known in the art, such as using a weight-for-age z-score and / or a height-for-age z-score.

[0556] mut-MMA

[0557] mut-MMA (MCID: MTH076, OMIM: 251000) is also known as methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency and is caused by various mutations in the gene MMUT encoding mitochondrial methylmalonyl-CoA mutase.

[0558] Mut-MMA can be derived from the complete (mut 0 Enzyme isoform) defective or partial (mut - OH-Cbl reactivity can be used to distinguish mut 0 and mut-subtypes (see e.g. F., et al., 2007. Pediatric research, 62(2), pp. 225-230). For example, cells from a patient that show at least a 1.5-fold increase in propionate incorporation after administration of OH-Cbl can be classified as mut - , whereas those with less or no increase can be classified as mut 0 .

[0559] In some embodiments, the MMA is mut-MMA and the MMA-associated polypeptide is MMUT or a fragment and / or variant thereof. The mut-MMA may be mut 0 Subtype or mut - In some embodiments, mut-MMA is mut 0 Subtype.

[0560] In one aspect, the present invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the present invention for use in preventing or treating mut-MMA.

[0561] In one aspect, the present invention provides use of the lentiviral vector, cell or pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating mut-MMA.

[0562] In one aspect, the present invention provides a method for preventing or treating mut-MMA, comprising administering a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0563] cblA-MMA

[0564] cblA-MMA (MCID: MTH077, OMIM: 251100) is also known as methylmalonic aciduria, cblA type, and is caused by multiple mutations in the gene MMAA encoding the mitochondrial methylmalonic aciduria type A protein.

[0565] In some embodiments, the MMA is cblA-MMA and the MMA-cognate polypeptide is MMAA or a fragment and / or variant thereof.

[0566] In one aspect, the present invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the present invention for use in preventing or treating cblA-MMA.

[0567] In one aspect, the present invention provides use of the lentiviral vector, cell or pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating cblA-MMA.

[0568] In one aspect, the present invention provides a method for preventing or treating cblA-MMA, comprising administering a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0569] cblB-MMA

[0570] cblB-MMA (MCID: MTH078, OMIM: 251110) is also known as methylmalonic aciduria, cblB type, and is caused by multiple mutations in the gene MMAB encoding the mitochondrial methylmalonic aciduria type B protein.

[0571] In some embodiments, the MMA is cblB-MMA and the MMA-associated polypeptide is MMAB or a fragment and / or variant thereof.

[0572] In one aspect, the present invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the present invention for use in preventing or treating cblB-MMA.

[0573] In one aspect, the present invention provides use of the lentiviral vector, cell or pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating cblB-MMA.

[0574] In one aspect, the present invention provides a method for preventing or treating cblB-MMA, comprising administering a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0575] cblD-MMA

[0576] cblD-MMA (MCID: MTH055, OMIM: 277410) is also known as methylmalonic aciduria and homocystinuria, cblD type, and is caused by multiple mutations in the gene MMADHC encoding the mitochondrial methylmalonic aciduria and homocystinuria type D protein.

[0577] In some embodiments, the MMA is cblD-MMA and the MMA-associated polypeptide is MMADHC or a fragment and / or variant thereof.

[0578] In one aspect, the present invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the present invention for use in preventing or treating cblD-MMA.

[0579] In one aspect, the present invention provides use of the lentiviral vector, cell or pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating cblD-MMA.

[0580] In one aspect, the present invention provides a method for preventing or treating cblD-MMA, comprising administering a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0581] MCEE Defects

[0582] MCEE deficiency (MCID: MTH040, OMIM: 251120) is also known as methylmalonyl-CoA epimerase deficiency and is caused by various mutations in the gene MCEE encoding the mitochondrial methylmalonyl-CoA epimerase.

[0583] In some embodiments, the MMA is MCEE-deficiency and the MMA-associated polypeptide is MCEE or a fragment and / or variant thereof.

[0584] In one aspect, the present invention provides a lentiviral vector, a cell or a pharmaceutical composition according to the present invention for use in preventing or treating MCEE deficiency.

[0585] In one aspect, the present invention provides use of a lentiviral vector, a cell or a pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating MCEE deficiency.

[0586] In one aspect, the present invention provides a method for preventing or treating MCEE deficiency, comprising administering a therapeutically effective amount of a lentiviral vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0587] Example

[0588] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0589] Example 1 - Lentiviral vector (LV) encoding MUT transgene

[0590] We generated and produced lentiviral vectors (LVs) encoding the MUT transgene and expressed MUT in vitro in hepatocyte cell lines and in wild-type (WT) mice and in the MMA mouse model. – / – ; Tg INS-MCK-Mut The lentiviral vector (LV) was evaluated in vivo in mice (MCK-Mut / -).

[0591] result

[0592] We generated and produced LVs encoding human wild-type or codon-optimized MUT transgenes under the control of a hepatocyte-specific cassette based on the enhanced transthyretin promoter (ET.MUT) or the human alpha-1 antitrypsin promoter (hAAT.MUT). Figure 1 a).

[0593] To compare between different MUT transgenes, we transduced Huh7 cells with LV-ET.MUTwt and LV-ET.MUTco_1 at increasing multiplicity of infection (MOI). As detected by Western blot analysis, we detected higher MUT expression in cells transduced with LV-ET.MUTco_1 compared to LV-ET.MUTwt ( Figure 1 b). The same results were obtained by transducing Huh7 cells with LV-hAAT.MUTwt or LV-hAAT.MUTco_1 at different MOIs ( Figure 1 c).

[0594] By MUT protein quantification and normalization to the GAPDH housekeeper, we demonstrated that, at similar vector DNA copies per cell (vector copy number, VCN), transgene expression was higher (approximately 2-fold) from expression cassettes driven by the ET promoter compared to hAAT and from codon-optimized transgenes compared to the wt version ( Figure 1 df).

[0595] To directly compare the in vivo liver transduction efficiency of LV-ET.MUTwt and LV-ET.MUTco, we isolated them at 2.5 × 10 10 The dose of transduction units (TU) / kg was administered to 2-week-old C57Bl / 6WT mice by iv injection. At the end of the experiment, 4 weeks after LV administration, the VCN in the liver was about 4 in the group injected with LV-ET.MUTwt and about 2 in the group injected with LV-ET.MUTco. Figure 2 a). Regarding mRNA expression in liver ( Figure 2 b) and protein export ( Figure 2 cd), the MUT transgene expression was higher in the group injected with LV-ET.MUTco compared to LV-ET.MUTwt.

[0596] We performed gene therapy experiments in MCK-Mut- / - mice, a model that exhibits clinical and biochemical features of MMA, including severe growth retardation, frailty, high serum MMA levels, and liver and kidney mitochondrial disease (Manoli, I., et al., 2018. JCI insight, 3(23), e12435). 10 Administration of LV-ET.MUTwt at a dose of 1.5 TU / kg into immature MCK-Mut- / - mice resulted in 100% survival and complete normalization of their growth curve ( Figure 3 a, b) and resulted in a significant reduction in circulating MMA levels until 12 months after LV administration ( Figure 3 c), thus demonstrating a significant therapeutic benefit of LV-mediated liver gene therapy.

[0597] Materials and methods

[0598] Plasmid construction

[0599] The MUT coding sequences (WT or codon-optimized) used in this study were synthesized by GeneScript and cloned into the third-generation self-inactivating (SIN) LV transfer plasmid (Milani, M., et al., 2019. Science Translational Medicine, 11(493), p.eaav7325) under the control of the enhanced transthyretin promoter (ET) or the human alpha-1 antitrypsin promoter (hAAT).

[0600] Vector production

[0601] Laboratory-grade VSV.G pseudotyped third generation SIN LVs were generated by transient calcium phosphate transfection into 293T cells or 293T MHC-negative CD47-high cells to obtain MHC-free CD47hi LVs. Cells were transfected with a solution containing a mixture of the selected LV genome transfer plasmid, packaging plasmids pMDLg / pRRE and pCMV.REV, pMD2.G and pAdvantage as described previously (Milani, M., et al., 2017. EMBO molecular medicine, 9(11), pp.1558-1573; and Milani, M., et al., 2019. Science Translational Medicine, 11(493), p.eaav7325).

[0602] The medium was replaced 14-16 hours after transfection, and the supernatant was collected about 30 hours after the medium change. The supernatant containing LV was sterilized by passing through a 0.22 μm filter (Millipore), transferred to a sterile polypoliallomer tube (Beckman), and centrifuged at 20,000 g for 120 minutes at 20°C (Beckman Optima XL-100K Ultracentrifuge). The LV pellet was resuspended in an appropriate volume of PBS to allow 500-1000X concentration.

[0603] LV titration

[0604] For LV titration, 1 × 10 cells were transduced with serial LV dilutions in the presence of polybrene (8 μg / ml). 5293T cells. 14 days after transduction, genomic DNA (gDNA) was extracted using the Maxwell 16 Cell DNA Purification Kit (Promega) according to the manufacturer's instructions. VCN was determined by ddPCR starting from 5-20 ng template gDNA using primers designed on the primer binding site region of LV (HIV fw: 5'-TACTGACGCTCTCGCACC-3'; HIV rv: 5'-TCTCGACGCAGGACTCG-3') and probe (FAM 5'-ATCTCTCTCCTTCTAGCCTC-3'). The amount of endogenous DNA was quantified by a primer / probe set designed on the human telomerase gene (Telo fw: 5'-GGCACACGTGGCTTTTCG-3'; Telo rv: 5'-GGTGAACCTCGTAAGTTTATGCAA-3'; Telo probe: VIC 5'-TCAGGACGTCGAGTGGACACGGTG-3'TAMRA) or human GAPDH gene (Applied Biosystems HS00483111_cm). PCR reactions were performed with each primer (900 nM) and probe (250 nM, 500 nM for Telo) according to the manufacturer's instructions (Biorad), read with a QX200 reader and analyzed with QuantaSoft software (Biorad). The infectious titer expressed as TU / mL was calculated using the formula U / mL = (VCN x 100,000 x (1 / dilution factor). LV physical particles were measured by HIV-1 Gag p24 antigen immunocapture assay (Perkin Elmer) according to the manufacturer's instructions. LV-specific infectivity was calculated as the ratio between infectious titer and physical particles.

[0605] Cell culture and in vitro transduction experiments

[0606] HuH7 cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA) at 37°C and 5% CO2. 5 Cells were harvested 10 days after LV transduction.

[0607] Mouse experiments

[0608] All animal experiments were performed in strict accordance with the Italian and European legislation on good animal practice (2010 / 63 / EU) on animal care and experimentation. Wild-type C57Bl / 6 or Mut - / - ; Tg INS-MCK-Mut 2-week-old mice. Via the retroorbital plexus at 2.5x10 10 Animals were administered LV at a dose of TU / kg. Blood samples were collected monthly throughout the study for analysis of metabolites (see "Blood MMA determination"). For experiments performed in WT C57Bl / 6 mice, animals were sacrificed 4 weeks after LV administration, and liver samples were collected and snap-frozen for vector copy number, mRNA, and protein analysis.

[0609] RNA analysis

[0610] RNA samples were extracted from mouse liver using Maxwell 16simplyRNA Tissue Kit (Promega). According to the manufacturer's instructions, cDNA was synthesized from 1 μg of total RNA using SuperScript IV VILO Master Mix (Invitrogen). Using primer / probe sets designed on the WPRE region of LV (WPRE: primer fw 5'-GGCTGTTGGGCACTGACAAT-3'; primer rv 5'-ACGTCCCGCGCAGAATC-3'; probe FAM 5'-TTTCCTTGGCTGCTCGCCTGTGT-3'NGB), LV gene expression was assessed by ddPCR from 25-50ng template cDNA. Mouse HPRT was used as a reference gene (Bio Rad, Mmu 10031256dMmu CPE5095493). PCR reactions were performed with each primer (900 nM) and probe (250 nM) according to the manufacturer's instructions (Biorad), read with a QX200 reader and analyzed with QuantaSoft software (Biorad).

[0611] Western blot analysis

[0612] Protein extracts from HuH7 cells or from mouse liver samples were prepared using RIPA buffer (EMD Millipore) and protease inhibitors (Roche). Protein concentration was determined using DC Protein Assay (Bio Rad Laboratories). SDS-page electrophoresis was performed in Bis-Tris 4-12% gradient polyacrylamide gels. After transfer, the membrane was blocked with 1XTBS+5% milk and incubated with anti-MUT antibody (Abcam ab67869) or anti-β-actin (Sigma AldrichA2228). The membrane was incubated with anti-mouse IgG secondary antibody (Jackson Immunoresearch 115-035-003), then incubated with clarity Western ECL substrate (Bio Rad) and visualized by Uvitec Imaging System (Cleaver Scientific). For protein blot quantification, we used Image J software.

[0613] Plasma and tissue MMA determination

[0614] Blood samples were collected via the retro-orbital plexus in test tubes filled with 0,5M EDTA. After withdrawal of the blood samples, dried blood spots (DBS) were obtained from the spot blood on filter paper (903; Whatman GmbH, Dassel, Germany). MMA measurements in DBS were performed by Triple-Quad Mass Spectrometry as previously described (Rizzo, C., et al., 2014. Clinica chimica acta, 429, pp. 30-33). Tissue MMA was determined in protein extracts from liver, kidney or brain samples and quantified by Triple-Quad Mass Spectrometry as described above.

[0615] VCN Determination

[0616] DNA was extracted from cells or liver samples using Maxwell 16Cell or Tissue DNA Purification Kits (Promega). VCN was determined in human macrophages as described above (see "LV titration"). VCN in mouse DNA was determined by ddPCR starting from 5-20 ng template gDNA using primer / probe sets designed on the primer binding site region of LV (see "LV titration" above). The amount of endogenous mouse DNA was quantified by a primer / probe set designed on the mouse Sema3A gene (Sema3A fw: 5'-ACCGATTCCAGATGATTGGC-3'; Sema3A rv: 5'-TCCATATTAATGCAGTGCTTGC-3'; Sema3A probe: HEX 5'-AGAGGCCTGTCCTGCAGCTCATGG-3'BHQ1). PCR reactions were performed with each primer (900 nM) and probe (250 nM) according to the manufacturer's instructions (Biorad), read with a QX200 reader and analyzed with QuantaSoft software (Biorad).

[0617] Transmission electron microscopy (TEM) analysis

[0618] For TEM analysis, tissues were fixed by needle injection perfusion with a 0.15 M fixative containing 2% formaldehyde, 2.5% glutaraldehyde in NaCacodylate buffer pH 7.4 as reported (Wisse E et al., World J Gastroenterol. 2010). After initial fixation, samples were left in the same fixative at 4°C overnight. The tissues were then cut into 1 mm 3The slides were post-fixed for 2 hours in 1% osmium tetroxide, 1.5% potassium ferricyanide in 100 mM NaCacodylate buffer on ice. The samples were dehydrated with crescent concentrations of ethanol, washed with propylene oxide and soaked overnight in a mixture of propylene oxide / epoxy resin. The resin was then replaced with fresh epoxy resin and the samples were embedded in silicone molds. Then, after curing at 60°C for 48 hours, the resin blocks were cut into ultrathin sections (70-90 nm) using an ultramicrotome (UC7, Leica microsystem, Vienna, Austria), collected on copper grids, stained with uranyl acetate and Sato's lead solution, and observed in a Transmission Electron Microscope Talos L120C (FEI, Thermo Fisher Scientific) operated at 120 kV. Images were acquired with a Ceta CCD camera (FEI, Thermo Fisher Scientific).

[0619] Liver collection for histopathological analysis

[0620] Livers were harvested from mice and fixed in buffered formalin by immersion. Livers were trimmed, embedded in paraffin, sectioned, mounted on glass slides and stained with hematoxylin and eosin (H&E).

[0621] Electron microscopic analysis of mitochondria in liver and kidney

[0622] For transmission electron microscopy analysis, tissues were fixed by needle injection (Wisse et al. (2010) World J Gastroenterol 16:2851-66) with a fixative containing 2% formaldehyde, 2.5% glutaraldehyde in 0.15M NaCacodylate buffer pH 7.4. After initial fixation, samples were left in the same fixative at 4°C overnight. The tissues were then cut into 1 mm 3The slides were post-fixed for 2 hours in 1% osmium tetroxide, 1.5% potassium ferricyanide in 100 mM NaCacodylate buffer on ice. The samples were dehydrated with crescent concentrations of ethanol, washed with propylene oxide and soaked overnight in a mixture of propylene oxide / epoxy resin. The resin was then replaced with fresh epoxy resin and the samples were embedded in silicone molds. Then, after curing at 60°C for 48 hours, the resin blocks were cut into ultrathin sections (70-90 nm) using an ultramicrotome (UC7, Leica microsystem, Vienna, Austria), collected on copper grids, stained with uranyl acetate and Sato's lead solution, and observed in a Transmission Electron Microscope Talos L120C (FEI, Thermo Fisher Scientific) operated at 120 kV. Images were acquired with a Ceta CCD camera (FEI, Thermo Fisher Scientific). For each tissue region corresponding to a single cell, 15 mitochondria were measured and 15 regions were measured, for a total of >200 mitochondria measured / mouse.

[0623] Statistical analysis

[0624] Statistical analysis was performed by Prism 9 software. When 2 or more independent groups were compared over time, two-way ANOVA was performed with Dunnet's multiple comparison test.

[0625] Example 2 - Long-term phenotypic correction of methylmalonic acidemia

[0626] We administered LV.ET-MUT to 2-week-old MCK-Mut- / - mice at a dose of 5E10 TU / kg and monitored them over a period of 12 months. LV treatment resulted in a complete rescue of the survival curve ( Figure 3 a) and normalization of growth rate, which was comparable to that of heterozygous littermates ( Figure 3 b). Importantly, we observed a rapid and significant reduction in circulating MMA compared to KO untreated controls, which was maintained long term (1 year after LV)( Figure 3 c). At the end of the experiment, we measured liver MMA, which was significantly reduced in MCK-Mut treated animals compared to KO untreated controls ( Figure 3 d).

[0627] By vector copy number (VCN) and mRNA analysis ( Figure 4 a and b) and MUT protein expression in tissue lysates ( Figure 4c), We demonstrated persistence of vector and MUT transgene expression in the liver of treated mice 12 months after LV administration.

[0628] Liver transduction efficiency was quantified by RNA ISH in two MCK-Mut treated mice and resulted in approximately 10-20% liver area ( Figure 5 ab). The data correlate with an overall correction of liver pathology in MCK-Mut mice, which were comparable to WT controls in terms of histopathological features ( Figure 5 c).

[0629] Electron microscopic analysis of MCKMut- / - livers confirmed the presence of giant, abnormally shaped mitochondria with shortened or absent cristae. LV-treated mice showed rescue of mitochondrial structure that was comparable to mitochondria from WT control animals ( Figure 6 a). Image quantification showing rescue of mitochondrial area, perimeter and shape measured as circularity index ( Figure 6 bd). Consistent with the correction of mitochondrial disease, the levels of toxic MMA in the liver were significantly reduced and almost normalized compared with KO untreated controls ( Figure 6 e).

[0630] In the proximal tubules of MCK-Mut- / - kidneys, mitochondria appeared disorganized, annular, and engulfed by cytosolic material. In LV-treated MCK-Mut- / - mice, there was no evidence of renal mitochondrial disease ( Figure 7 a). Image quantification showed that the number of ring structures was comparable to that of WT control mice ( Figure 7 b). Consistent with correction for mitochondrial disease, levels of toxic MMA in the kidneys were significantly reduced compared with KO untreated controls ( Figure 7 c).

[0631] Electron microscopic analysis of the cerebral cortex of MCK-Mut- / - mice revealed some minor changes in mitochondrial structure. LV-treated mice showed mitochondrial ultrastructure comparable to that of WT control animals ( Figure 8 a). Regarding MMA in brain tissue, we show accumulation of toxic metabolites in MCK-Mut- / - untreated animals and a 3-fold reduction in brain MMA in LV-treated mice, which is mediated by hepatic detoxification ( Figure 8 b).

[0632] Administration of LV.ET-MUT to immature MCK-Mut- / - mice resulted in a complete rescue of survival and growth curves and in a long-term significant reduction in circulating MMA levels as well as liver, kidney, and brain MMA, with amelioration of hepatorenal mitochondrial disease.

[0633] The reduction in circulating MMA and rescue of growth curves were confirmed in gene therapy experiments in which CD47 was administered at a dose of 5E10 TU / kg to immature MCK-Mut- / - mice. 高 h-MCH 无 LV.ET-MUT( Fig. 9 ).

[0634] Administration of LV.ET-MUTco to immature MCK-Mut- / - mice resulted in a rescue of the growth curve that was similar or slightly improved compared to LV.ET-MUTco at the same LV dose (2.5E10 TU / kg), as well as a reduction in circulating MMA ( Fig.10 ).

[0635] Example 3 - Lentiviral vector (LV) encoding BSEP transgene

[0636] We generated a lentiviral vector (LV) encoding the BSEP transgene and evaluated it in vitro in a hepatocyte cell line and in vivo in a mouse model of PFIC-2 (Abcb11- / - mice).

[0637] result

[0638] We generated and produced LVs encoding human wild-type or codon-optimized BSEP transgenes under the control of a hepatocyte-specific cassette based on the enhanced transthyretin promoter (ET.BSEP) or the human alpha-1 antitrypsin promoter (hAAT.BSEP). Fig.11 a).

[0639] To make comparisons between different BSEP transgenes, we first transduced Huh7 cells with LV-ET.BSEPwt, LV-ET.BSEPco_1 or LV-ET.BSEPco_2 at increasing multiplicity of infection (MOI). We performed flow cytometric analysis 10 days after transduction, which detected BSEP overexpression in LV-ET.BSEP-transduced cells. The mean fluorescence intensity increased proportionally to the MOI employed, and at similar vector copy numbers per cell (VCN), the mean fluorescence intensity of BSEPco_1 and BSEPco_2 transgenes was higher compared to BSEPwt ( Fig.11 b, c).

[0640] We then compared the activities of the ET and hAAT promoters by transducing Huh7 cells with LV-ET.BSEPco_1 or LV-hAAT.BSEP_co_1. VCN / cell was comparable in cells transduced with both vectors at different MOIs ( Fig.11d), while BSEP protein expression was higher in LV-ET.BSEPco_1 compared with LV-hAAT.BSEPco_1 transduced cells as detected by Western blot analysis ( Fig.11 e). By BSEP protein quantification and normalization to the GAPDH housekeeper, we confirmed that transgene expression was higher (3-3.5-fold) from expression cassettes driven by the ET promoter compared to hAAT ( Fig.11 f).

[0641] Immunofluorescence analysis on Huh7 cells transduced with LV-ET.BSEPco_1 confirmed proper BSEP membrane and cytoplasmic localization, whereas no signal was detected in Huh7 non-transduced cells ( Fig.12 ).

[0642] We use 3x10 10 LV-ET.BSEPco_1 was administered to juvenile Abcb11- / - mice by iv injection at a dose of 100 transduction units (TU) / kg. Treatment resulted in a significant decrease in serum BA, bilirubin, and ALP biomarkers until 7 months of age, suggesting prevention of progressive cholestatic injury ( Fig.13 ac).

[0643] Materials and methods

[0644] Plasmid construction

[0645] The BSEP coding sequences (WT or codon-optimized) used in this study were synthesized by GeneScript and cloned into the third-generation self-inactivating (SIN) LV transfer plasmid (Milani, M., et al., 2019. Science Translational Medicine, 11(493), p.eaav7325) under the control of the enhanced transthyretin promoter (ET) or the human alpha-1 antitrypsin promoter (hAAT).

[0646] Vector production

[0647] Laboratory-grade VSV.G pseudotyped third generation SINLV was produced by transient calcium phosphate transfection into 293T cells. As previously described, 293T cells were transfected with a solution containing a mixture of the selected LV genome transfer plasmid, packaging plasmids pMDLg / pRRE and pCMV.REV, pMD2.G and pAdvantage (Milani, M., et al., 2017. EMBO molecular medicine, 9 (11), pp. 1558-1573; and Milani, M., et al., 2019. Science Translational Medicine, 11 (493), p.eaav7325).

[0648] The medium was replaced 14-16 hours after transfection, and the supernatant was collected about 30 hours after the medium change. The supernatant containing LV was sterilized by passing through a 0.22 μm filter (Millipore), transferred to a sterile polypoliallomer tube (Beckman), and centrifuged at 20,000 g for 120 minutes at 20°C (Beckman Optima XL-100K Ultracentrifuge). The LV pellet was resuspended in an appropriate volume of PBS to allow 500-1000X concentration.

[0649] LV titration

[0650] For LV titration, 1 × 10 cells were transduced with serial LV dilutions in the presence of polybrene (8 μg / ml). 5293T cells.10 days after transduction, Maxwell 16Cell DNA Purification Kit (Promega) was used to extract genomic DNA (gDNA) according to the manufacturer's instructions. Using primers (HIV fw: 5'-TACTGACGCTCTCGCACC-3' designed on the primer binding site region of LV; HIV rv: 5'-TCTCGACGCAGGACTCG-3') and probe (FAM 5'-ATCTCTCTCCTTCTAGCCTC-3'), VCN was determined from 5-20ng template gDNA by digital droplet PCR (ddPCR). The amount of endogenous DNA was quantified by the primer / probe set (Applied Biosystems HS00483111_cm) designed on the human GAPDH gene. PCR reaction was carried out with each primer (900nM) and probe (250nM) according to the manufacturer's instructions (Biorad), read with QX200 reader and analyzed with QuantaSoft software (Biorad). The infectious titer expressed as TU / mL was calculated using the formula U / mL = (VCN x 100,000 x (1 / dilution factor)). LV physical particles were measured by HIV-1 Gag p24 antigen immunocapture assay (Perkin Elmer) according to the manufacturer's instructions. LV specific infectivity was calculated as the ratio between infectious titer and physical particles.

[0651] Cell culture and in vitro transduction experiments

[0652] HuH7 cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA) at 37°C and 5% CO2. 5 Cells were placed in 100 μL of LV per well (100 μL / well) and transduced with LV variants at different MOIs in the presence of polybrene (8 μg / ml). Ten days after LV transduction, cells were harvested for molecular analysis.

[0653] Mouse experiments

[0654] All animal experiments were performed in strict accordance with the Italian and European legislation on animal care and experimentation (2010 / 63 / EU) for good animal practice. 2-week-old Abcb11- / - mice (Jackson strain n.004125) were used in these studies. 10Animals were administered a dose of TU / kg using LV. Blood samples were collected monthly throughout the study for metabolite analysis.

[0655] VCN Determination

[0656] DNA was extracted from cells or liver samples using Maxwell 16Cell or Tissue DNA Purification Kits (Promega). VCN was determined in Huh7 samples as described above (see "LV titration"). VCN in mouse DNA was determined by ddPCR starting from 5-20 ng template gDNA using primer / probe sets designed on the primer binding site region of LV (see "LV titration" above). The amount of endogenous mouse DNA was quantified by a primer / probe set designed on the mouse Sema3A gene (Sema3A fw: 5'-ACCGATTCCAGATGATTGGC-3'; Sema3A rv: 5'-TCCATATTAATGCAGTGCTTGC-3'; Sema3A probe: HEX 5'-AGAGGCCTGTCCTGCAGCTC ATGG-3'BHQ1).

[0657] PCR reactions were performed with each primer (900 nM) and probe (250 nM) according to the manufacturer's instructions (Biorad), read with a QX200 reader and analyzed with QuantaSoft software (Biorad).

[0658] Western blot analysis

[0659] Protein extracts from HuH7 cells or from mouse liver samples were prepared using RIPA buffer (EMD Millipore) and protease inhibitors (Roche). Protein concentration was determined using DC Protein Assay (Bio Rad Laboratories). SDS-page electrophoresis was performed in Bis-Tris 4-12% gradient polyacrylamide gels. After transfer, the membrane was blocked with 1XTBS+5% milk and incubated with anti-Abcb11 antibody (Sigma-Aldrich HPA019035) or anti-GAPDH (Biolegend 649201). The membrane was incubated with anti-mouse and anti-rabbit IgG secondary antibodies (Jackson Immunoresearch115-035-003 and 111-035-144), then incubated with clarity Western ECL substrate (Bio Rad) and visualized by Uvitec Imaging System (Cleaver Scientific). For protein blot quantification, we used Image J software.

[0660] BSEP immunofluorescence analysis

[0661] Huh7 LV-transduced cells were plated in 8-well slides ( ChamberSlide TM system) to reach 40-50% confluence. After 24 hours, cells were fixed in 4% PFA and permeabilized in PBS+Triton X-100 0.1% at room temperature (RT) for 10 minutes. Cells were blocked in PBST+1% BSA for 1 hour at RT and stained overnight with rabbit anti-BSEP (ab255605, diluted 1:100) primary antibody at 4°C. After incubation with goat anti-rabbit IgG 488 secondary antibody (Invitrogen, dil.1:1000), nuclear staining was performed with Hoechst (diluted at 2ug / mL in PBS) at RT for 5 minutes. Stained slides were acquired under a Leica TCS SP8 SMD confocal microscope.

[0662] Bile acid, ALP, bilirubin determination

[0663] Total bile acids in serum samples were determined by using Mouse Total Bile Acid Assay Kit (CrystalChem, 80471) according to the manufacturer's instructions. Serum ALP activity was measured in a SABA chemistry analyzer (SEAC-Radim) using a kinetic UV method optimized by the International Federation of Clinical Chemistry and Laboratory Medicine, and was expressed as units per liter (U / L).

[0664] Example 4 - Lentiviral vector (LV) encoding DBT transgene

[0665] We generated and produced lentiviral vectors (LVs) encoding a DBT transgene encoding the E2 subunit of BCKDC and evaluated them in vitro in a hepatocyte cell line and in vivo in both wild-type (WT) mice and iMSUD mice, an intermediate mouse model of MSUD caused by E2 deficiency.

[0666] result

[0667] We generated and produced LVs carrying human wild-type or codon-optimized DBT transgenes under the control of a hepatocyte-specific cassette based on the enhanced transthyretin promoter (ET.DBT) or the human alpha-1 antitrypsin promoter (hAAT.DBT) ( Fig.14 a).

[0668] To compare between the different DBT transgenes, we transduced Huh7 cells with LV-ET.DBTwt and LV-ET.DBTco_1 at increasing multiplicity of infection (MOI). As detected by Western blot analysis, we detected higher DBT expression in cells transduced with LV-ET.DBTco_1 compared to LV-ET.DBTwt ( Fig.14 b). The same results were obtained by transducing Huh7 cells with LV-hAAT.DBTwt or LV-hAAT.DBTco_1 at different MOIs ( Fig.14 c).

[0669] By DBT protein quantification and normalization to the GAPDH housekeeper, we demonstrated that, at similar vector DNA copies per cell (vector copy number, VCN), transgene expression was higher (approximately 7-8 fold) from expression cassettes driven by the ET promoter compared to hAAT and from codon-optimized transgenes compared to the wt version ( Fig.14 df).

[0670] To directly compare the in vivo liver transduction efficiency of LV-ET.DBTwt and LV-ET.DBTco, we isolated them at 2.5 × 10 10 The dose of transduction units (TU) / kg was administered to immature C57Bl / 6WT mice by iv injection. At the end of the experiment, 4 weeks after LV administration, the vector copy number (VCN) in the liver of both treatment groups was approximately 0.4 ( Fig.15 a). DBT mRNA was slightly higher in the group injected with LV-ET.DBTwt, while DBT protein output in the liver of the LV-ET.DBTco group was slightly higher ( Fig.15 bd).

[0671] We use 5x10 10 LV-ET.DBTco_1 was administered to immature mice by iv injection at a dose of 100 transducing units (TU) / kg. Treatment resulted in improved survival in treated mice (50% survival probability at day 35 and day 111, respectively) compared to knockout littermates ( Fig.16 a). Measurements of circulating BCAA and alloisoleucine showed that these biomarkers remained stable in LV-treated animals until week 12 after LV ( Fig.16 b, c) At the last time point, these parameters could not be evaluated in iMSUD-untreated animals that had previously died.

[0672] Materials and methods

[0673] Plasmid construction

[0674] The DBT coding sequences (WT or codon-optimized) used in this study were synthesized by GeneScript and cloned into the third-generation self-inactivating (SIN) LV transfer plasmid (Milani, M., et al., 2019. Science Translational Medicine, 11(493), p.eaav7325) under the control of the enhanced transthyretin promoter (ET) or the human alpha-1 antitrypsin promoter (hAAT).

[0675] Vector production

[0676] Laboratory-grade VSV.G pseudotyped third generation SINLV was produced by transient calcium phosphate transfection into 293T cells. As previously described, 293T cells were transfected with a solution containing a mixture of the selected LV genome transfer plasmid, packaging plasmids pMDLg / pRRE and pCMV.REV, pMD2.G and pAdvantage (Milani, M., et al., 2017. EMBO molecular medicine, 9 (11), pp. 1558-1573; and Milani, M., et al., 2019. Science Translational Medicine, 11 (493), p.eaav7325).

[0677] The medium was replaced 14-16 hours after transfection, and the supernatant was collected about 30 hours after the medium change. The supernatant containing LV was sterilized by passing through a 0.22 μm filter (Millipore), transferred to a sterile polypoliallomer tube (Beckman), and centrifuged at 20,000 g for 120 minutes at 20°C (Beckman Optima XL-100K Ultracentrifuge). The LV pellet was resuspended in an appropriate volume of PBS to allow 500-1000X concentration.

[0678] LV titration

[0679] For LV titration, 1 × 10 cells were transduced with serial LV dilutions in the presence of polybrene (8 μg / ml). 5293T cells.10 days after transduction, Maxwell 16Cell DNA Purification Kit (Promega) was used to extract genomic DNA (gDNA) according to the manufacturer's instructions. Using primers (HIV fw: 5'-TACTGACGCTCTCGCACC-3' designed on the primer binding site region of LV; HIV rv: 5'-TCTCGACGCAGGACTCG-3') and probe (FAM 5'-ATCTCTCTCCTTCTAGCCTC-3'), VCN was determined from 5-20ng template gDNA by digital droplet PCR (ddPCR). The amount of endogenous DNA was quantitatively measured by the primer / probe group (Applied Biosystems HS00483111_cm) designed on the human GAPDH gene. According to the manufacturer's instructions (Biorad), PCR reaction was carried out with each primer (900nM) and probe (250nM), read with QX200 reader and analyzed with QuantaSoft software (Biorad). The infectious titer expressed as TU / mL was calculated using the formula U / mL = (VCN x 100000 x (1 / dilution factor)). LV physical particles were measured by HIV-1 Gagp24 antigen immunocapture assay (Perkin Elmer) according to the manufacturer's instructions. LV specific infectivity was calculated as the ratio between infectious titer and physical particles.

[0680] Cell culture and in vitro transduction experiments

[0681] HuH7 cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA) at 37°C and 5% CO2. 5 Cells were placed in 100 μL of LV per well (100 μL / well) and transduced with LV variants at different MOIs in the presence of polybrene (8 μg / ml). Ten days after LV transduction, cells were harvested for molecular analysis.

[0682] Mouse experiments

[0683] All animal experiments were performed in strict accordance with the good animal practice of Italian and European legislation on animal care and experimentation (2010 / 63 / EU). Wild-type C57Bl / 6 or iMSUD immature mice were used in these studies. iMSUD is a moderate MSUD mouse model that is DBT KO and shows low activity of BCKAD in the liver (Homanics, GE, et al., 2006. BMC medical genetics, 7 (1), pp. 1-13). The rats were injected with 2.5-5x10 10 Animals were administered LV at a dose of TU / kg. Blood samples were collected monthly throughout the study for metabolite analysis. For experiments performed in WT C57Bl / 6 mice, animals were sacrificed 4 weeks after LV administration, and liver samples were collected and snap-frozen for vector copy number, mRNA, and protein analysis.

[0684] RNA analysis

[0685] RNA samples were extracted from mouse liver using Maxwell 16simplyRNA Tissue Kit (Promega). According to the manufacturer's instructions, cDNA was synthesized from 1 μg of total RNA using SuperScript IV VILO Master Mix (Invitrogen). Using primer / probe sets designed on the WPRE region of LV (WPRE: primer fw 5'-GGCTGTTGGGCACTGACAAT-3'; primer rv 5'-ACGTCCCGCGCAGAATC-3'; probe FAM 5'-TTTCCTTGGCTGCTCGCCTGTGT-3'NGB), LV gene expression was assessed by ddPCR from 25-50ng template cDNA. Mouse HPRT was used as a reference gene (Bio Rad, Mmu 10031256dMmu CPE5095493). PCR reactions were performed with each primer (900 nM) and probe (250 nM) according to the manufacturer's instructions (Biorad), read with a QX200 reader and analyzed with QuantaSoft software (Biorad).

[0686] VCN Determination

[0687] DNA was extracted from cells or liver samples using Maxwell 16Cell or Tissue DNA Purification Kits (Promega). VCN was determined in Huh7 samples as described above (see "LV titration"). VCN in mouse DNA was determined by ddPCR starting from 5-20 ng template gDNA using primer / probe sets designed on the primer binding site region of LV (see "LV titration" above). The amount of endogenous mouse DNA was quantified by a primer / probe set designed on the mouse Sema3A gene (Sema3A fw: 5'-ACCGATTCCAGATGATTGGC-3'; Sema3A rv: 5'-TCCATATTAATGCAGTGCTTGC-3'; Sema3A probe: HEX 5'-AGAGGCCTGTCCTGCAGCTC ATGG-3'BHQ1).

[0688] PCR reactions were performed with each primer (900 nM) and probe (250 nM) according to the manufacturer's instructions (Biorad), read with a QX200 reader and analyzed with QuantaSoft software (Biorad).

[0689] Western blot analysis

[0690] Protein extracts from HuH7 cells or from mouse liver samples were prepared using RIPA buffer (EMD Millipore) and protease inhibitors (Roche). Protein concentration was determined using DC Protein Assay (Bio Rad Laboratories). SDS-page electrophoresis was performed in Bis-Tris 4-12% gradient polyacrylamide gels. After transfer, the membrane was blocked with 1XTBS+5% milk and incubated with anti-DBT antibody (Sigma Aldrich HPA026481) or anti-β-actin (Sigma Aldrich A2228). The membrane was incubated with anti-mouse and anti-rabbit IgG secondary antibodies (Jackson Immunoresearch 115-035-003 and 111-035-144), then incubated with clarity Western ECL substrate (Bio Rad) and visualized by Uvitec Imaging System (Cleaver Scientific). For protein blot quantification, we used Image J software.

[0691] Blood BCAA and alloisoleucine determination

[0692] Blood samples were collected via the retro-orbital plexus in tubes filled with 0,5 M EDTA. After withdrawal of blood samples, dried blood spots (DBS) were obtained from spotted blood on filter paper (903; Whatman GmbH, Dassel, Germany). BCAA and alloisoleucine measurements in DBS were performed by liquid chromatography-tandem mass spectrometry as described previously.

[0693] Statistical analysis

[0694] Statistical analysis was performed by using Prism 9 software. Comparison of survival curves was performed by applying the Long-rank (Mantel-Cox) test.

[0695] Implementation

[0696] Various preferred features and embodiments of the invention will now be described with reference to the following numbered paragraphs.

[0697] 1. A lentiviral vector comprising a nucleotide sequence encoding a methylmalonic acidemia (MMA)-associated polypeptide, optionally wherein the lentiviral vector is an immune-shielded lentiviral vector.

[0698] 2. A lentiviral vector according to paragraph 1, wherein the MMA-associated polypeptide is selected from methylmalonyl-CoA mutase (MMUT) or a fragment thereof; methylmalonic aciduria type A (MMAA) or a fragment thereof; methylmalonic aciduria type B (MMAB) or a fragment thereof; methylmalonic aciduria and homocystinuria type D (MMADHC) or a fragment thereof; or methylmalonyl-CoA epimerase (MCEE) or a fragment thereof.

[0699] 3. The lentiviral vector according to paragraph 1 or 2, wherein the MMA-associated polypeptide is MMUT or a fragment thereof.

[0700] 4. A lentiviral vector according to any of the preceding paragraphs, wherein the MMA-associated polypeptide comprises or consists of an amino acid sequence that is at least 70% identical to one of SEQ ID NO: 37, 40, 42, 44 or 46, or a fragment thereof, preferably, wherein the MMA-associated polypeptide comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 37, or a fragment thereof.

[0701] 5. A lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of a nucleotide sequence at least 70% identical to one of SEQ ID NO: 38, 39, 41, 43, 45 or 47, or a fragment thereof, preferably, wherein the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of a nucleotide sequence at least 70% identical to one of SEQ ID NO: 38 or 39, or a fragment thereof.

[0702] 6. A lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide is codon-optimized, preferably, wherein the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 39 or a fragment thereof.

[0703] 7. The lentiviral vector according to any one of the preceding paragraphs, wherein the lentiviral vector is a CD47 高 Lentiviral vectors.

[0704] 8. The lentiviral vector according to any one of the preceding paragraphs, wherein the lentiviral vector is obtained from CD47 高 A host cell, optionally wherein the host cell is genetically engineered to increase expression of CD47 on the cell surface.

[0705] 9. The lentiviral vector according to any of the preceding paragraphs, wherein the lentiviral vector has at least about 2-fold more CD47 on its surface than a lentiviral vector obtained from an unmodified host cell.

[0706] 10. The lentiviral vector according to any one of the preceding paragraphs, wherein the lentiviral vector is an MHC-I 无 Lentiviral vectors.

[0707] 11. A lentiviral vector according to any one of the preceding paragraphs, wherein the lentiviral vector is obtained from MHC-I 无 A host cell, optionally wherein the host cell is genetically engineered to disrupt expression of MHC-I on the cell surface.

[0708] 12. The lentiviral vector according to any of the preceding paragraphs, wherein MHC-I is undetectable on the surface of the lentiviral vector.

[0709] 13. The lentiviral vector according to any one of the preceding paragraphs, wherein the lentiviral vector is a CD47 高 / MHC-I 无 Lentiviral vectors.

[0710] 14. The lentiviral vector according to any one of the preceding paragraphs, wherein the lentiviral vector is obtained from CD47高 / MHC-I 无 Host cell.

[0711] 15. The lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to one or more miRNA target sequences.

[0712] 16. The lentiviral vector according to paragraph 15, wherein the one or more miRNA target sequences inhibit transgene expression in one or more cells other than hepatocytes, preferably wherein the one or more miRNA target sequences inhibit transgene expression in hematopoietic lineage cells and / or antigen presenting cells.

[0713] 17. The lentiviral vector according to paragraph 15 or 16, wherein the one or more miRNA target sequences are selected from miR-181, miR-142, miR-223 and miR-155 target sequences.

[0714] 18. The lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to one or more mir-142 target sequences, two or more mir-142 target sequences, three or more mir-142 target sequences, or four or more mir-142 target sequences.

[0715] 19. The lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to four mir-142 target sequences.

[0716] 20. A lentiviral vector according to any one of paragraphs 15-19, wherein the one or more miRNA target sequences comprise or consist of a nucleotide sequence at least 90% identical to SEQ ID NO: 17, preferably, wherein the one or more miRNA target sequences comprise or consist of a nucleotide sequence at least 90% identical to SEQ ID NO: 18.

[0717] 21. The lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a liver-specific promoter, preferably wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a hepatocyte-specific promoter.

[0718] 22. The lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a transthyretin (TTR) promoter, an alpha-1-antitrypsin (AAT) promoter, a thyroxine-binding globulin (TBG) promoter, an APoE / hAAT promoter, an HCR-hAAT promoter, an LP1 promoter, or an HLP promoter.

[0719] 23. The lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a transthyretin (TTR) promoter, preferably wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to an Enh1mTTR (ET) promoter.

[0720] 24. The lentiviral vector according to any of the preceding paragraphs, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a promoter comprising or consisting of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 19.

[0721] 25. The lentiviral vector according to any of the preceding paragraphs, wherein the lentiviral vector is pseudotyped, preferably wherein the lentiviral vector is VSV.G pseudotyped.

[0722] 26. A lentiviral vector according to any of the preceding paragraphs, wherein the lentiviral vector is a self-inactivating (SIN) lentiviral vector, preferably wherein the lentiviral vector comprises a self-inactivating (SIN) LTR, the LTR comprising or consisting of a nucleotide sequence at least 70% identical to SEQ ID NO: 23 or a fragment thereof.

[0723] 27. The lentiviral vector according to any of the preceding paragraphs, wherein the lentiviral vector is an integrating lentiviral vector and / or a replication-defective lentiviral vector.

[0724] 28. The lentiviral vector according to any of the preceding paragraphs, wherein the lentiviral vector comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO:36.

[0725] 29. An isolated cell comprising a lentiviral vector according to any one of paragraphs 1-28.

[0726] 30. A pharmaceutical composition comprising a lentiviral vector according to any one of paragraphs 1-28 in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0727] 31. A lentiviral vector according to any one of paragraphs 1 to 28 or a pharmaceutical composition according to paragraph 30 for use as a medicament.

[0728] 32. Use of a lentiviral vector according to any one of paragraphs 1 to 28 or a pharmaceutical composition according to paragraph 30 for the preparation of a medicament.

[0729] 33. A method comprising administering to a subject in need thereof a therapeutically effective amount of a lentiviral vector according to any one of paragraphs 1-28 or a pharmaceutical composition according to paragraph 30.

[0730] 34. A lentiviral vector according to any one of paragraphs 1 to 28 or a pharmaceutical composition according to paragraph 30 for use in preventing or treating methylmalonic acidemia (MMA).

[0731] 35. Use of the lentiviral vector according to any one of paragraphs 1 to 28 or the pharmaceutical composition according to paragraph 30 for the preparation of a medicament for preventing or treating methylmalonic acidemia (MMA).

[0732] 36. A method for preventing or treating methylmalonic acidemia (MMA), comprising administering to a subject in need thereof a therapeutically effective amount of a lentiviral vector according to any one of paragraphs 1-28 or a pharmaceutical composition according to paragraph 30.

[0733] 37. A lentiviral vector or pharmaceutical composition for use according to paragraph 34, the use according to paragraph 35, or the method according to paragraph 36, wherein the MMA-associated polypeptide is MMUT or a fragment thereof, and MMA is mut-type MMA (mut-MMA); wherein the MMA-associated polypeptide is MMAA or a fragment thereof, and MMA is cblA-type MMA (cblA-MMA); wherein the MMA-associated polypeptide is MMAB or a fragment thereof, and MMA is cblB-type MMA (cblB-MMA); wherein the MMA-associated polypeptide is MMADHC or a fragment thereof, and MMA is cblD-type MMA (cblD-MMA); or wherein the MMA-associated polypeptide is MCEE or a fragment thereof, and MMA is due to MCEE deficiency.

[0734] 38. A lentiviral vector or pharmaceutical composition for use according to paragraph 34 or 37, a use according to paragraph 35 or 37, or a method according to paragraph 36 or 37, wherein the MMA-associated polypeptide is MMUT or a fragment thereof, and MMA is mut-MMA, preferably wherein mut-MMA is mut 0 isoform or mut-isoform.

[0735] 39. The lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 31, 34, 37-38, the use according to any one of paragraphs 32, 35, 37-38, or the method according to any one of paragraphs 33, 36-39, wherein the subject is a human subject.

[0736] 40. A lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 31, 34, 37-39, a use according to any one of paragraphs 32, 35, 37-39, or a method according to any one of paragraphs 33, 36-39, wherein the subject is a minor.

[0737] 41. The lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 31, 34, 37-39, the use according to any one of paragraphs 32, 35, 37-39, or the method according to any one of paragraphs 33, 36-39, wherein the subject is a pediatric patient, preferably wherein the subject is a neonatal patient or an infant patient.

[0738] 42. The lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 31, 34, 37-41, the use according to any one of paragraphs 32, 35, 37-41, or the method according to any one of paragraphs 33, 36-41, wherein the lentiviral vector or the pharmaceutical composition is administered systemically, preferably wherein the lentiviral vector or the pharmaceutical composition is administered by intravenous injection or intraperitoneal injection.

[0739] 43. The lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 31, 34, 37-41, the use according to any one of paragraphs 32, 35, 37-41, or the method according to any one of paragraphs 33, 36-41, wherein the lentiviral vector or the pharmaceutical composition is administered locally, preferably wherein the lentiviral vector or the pharmaceutical composition is administered by direct injection, intra-arterial injection or intra-portal vein injection.

[0740] 44. The lentiviral vector or the pharmaceutical composition for use according to paragraph 43, the use according to paragraph 43 or the method according to paragraph 43, wherein the lentiviral vector or the pharmaceutical composition is administered locally to the liver, preferably wherein the lentiviral vector or the pharmaceutical composition is administered by intrahepatic injection, intrahepatic arterial injection or intraportal vein injection.

[0741] 45. A lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 31, 34, 37-44, a use according to any one of paragraphs 32, 35, 37-43, or a method according to any one of paragraphs 33, 36-44, wherein the lentiviral vector is expressed in an amount of at least about 10 8 TU / kg, at least about 10 9 TU / kg, or at least about 10 10 The dose of TU / kg was administered.

[0742] 46. ​​A lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 31, 34, 37-45, a use according to any one of paragraphs 32, 35, 37-45, or a method according to any one of paragraphs 33, 36-45, wherein the lentiviral vector is expressed in an amount of about 10 8 to about 1011TU / kg, from about 10 8 to about 10 10 TU / kg, or about 10 9 to about 1010 The dose of TU / kg was administered.

[0743] 47. A lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 31, 34, 37-46, a use according to any one of paragraphs 32, 35, 37-46, or a method according to any one of paragraphs 33, 36-46, wherein the lentiviral vector integrates into the genome of hepatocytes and is maintained as a hepatocyte replica, preferably, wherein the lentiviral vector integrates into the genome of hepatocytes and is maintained as a hepatocyte replica.

[0744] 48. A lentiviral vector or pharmaceutical composition for use according to any of paragraphs 34, 37-47, a use according to any of paragraphs 35, 37-47, or a method according to any of paragraphs 36-47, wherein plasma methylmalonic acid levels and / or urine methylmalonic acid levels are reduced and / or normalized.

[0745] 49. The lentiviral vector or pharmaceutical composition for use according to any one of paragraphs 34, 37-48, the use according to any one of paragraphs 35, 37-48, or the method according to any one of paragraphs 36-48, wherein mitochondrial function is improved, preferably wherein the plasma level of Fgf21 is reduced.

[0746] All disclosures mentioned in the above description are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the disclosed vectors, cells, compositions, test kits and uses of the present invention will be apparent to those skilled in the art. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications to the disclosed modes for implementing the present invention that are apparent to those skilled in the art are intended to be within the scope of the appended claims.

Claims

1. An immune-shielded lentiviral vector comprising a nucleotide sequence encoding a methylmalonic acidemia (MMA)-associated polypeptide.

2. The lentiviral vector according to claim 1, wherein the MMA-associated polypeptide is selected from methylmalonyl-CoA mutase (MMUT) or a fragment thereof; methylmalonic aciduria type A (MMAA) or a fragment thereof; methylmalonic aciduria type B (MMAB) or a fragment thereof; methylmalonic aciduria and homocystinuria type D (MMADHC) or a fragment thereof; or methylmalonyl-CoA isomerase (MCEE) or a fragment thereof.

3. The lentiviral vector according to claim 1 or 2, wherein the MMA-associated polypeptide is MMUT or a fragment thereof.

4. The lentiviral vector according to any one of the preceding claims, wherein the MMA-associated polypeptide comprises or consists of: An amino acid sequence at least 70% identical to one of SEQ ID NO:37, 40, 42, 44 or 46, or a fragment thereof, preferably, wherein the MMA-associated polypeptide comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO:37, or a fragment thereof.

5. The lentiviral vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of: A nucleotide sequence at least 70% identical to one of SEQ ID NO: 38, 39, 41, 43, 45 or 47, or a fragment thereof, preferably, wherein the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of a nucleotide sequence at least 70% identical to one of SEQ ID NO: 38 or 39, or a fragment thereof.

6. The lentiviral vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide is codon-optimized, preferably, wherein the nucleotide sequence encoding the MMA-associated polypeptide comprises or consists of: A nucleotide sequence at least 70% identical to SEQ ID NO: 39 or a fragment thereof.

7. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is a CD47 高 Lentiviral vectors.

8. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is obtained from CD47 高 A host cell, optionally wherein the host cell is genetically engineered to increase expression of CD47 on the surface of the cell.

9. The lentiviral vector of any of the preceding claims, wherein the lentiviral vector has at least about 2-fold more CD47 on its surface than a lentiviral vector obtained from an unmodified host cell.

10. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is an MHC-I 无 Lentiviral vectors.

11. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is obtained from MHC-I 无 A host cell, optionally wherein the host cell is genetically engineered to disrupt expression of MHC-I on the surface of the cell.

12. The lentiviral vector of any of the preceding claims, wherein MHC-I is undetectable on the surface of the lentiviral vector.

13. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is a CD47 高 / MHC-I 无 Lentiviral vectors.

14. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is obtained from CD47 高 / MHC-I 无 Host cell.

15. The lentiviral vector of any one of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to one or more miRNA target sequences.

16. The lentiviral vector according to claim 15, wherein the one or more miRNA target sequences inhibit transgene expression in one or more cells other than hepatocytes, preferably wherein the one or more miRNA target sequences inhibit transgene expression in hematopoietic lineage cells and / or antigen presenting cells.

17. The lentiviral vector according to claim 15 or 16, wherein the one or more miRNA target sequences are selected from miR-181, miR-142, miR-223 and miR-155 target sequences.

18. The lentiviral vector of any of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to one or more mir-142 target sequences, two or more mir-142 target sequences, three or more mir-142 target sequences, or four or more mir-142 target sequences.

19. The lentiviral vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to four mir-142 target sequences.

20. The lentiviral vector according to any one of claims 15 to 19, wherein the one or more miRNA target sequences comprise or consist of: A nucleotide sequence at least 90% identical to SEQ ID NO: 17, preferably, wherein the one or more miRNA target sequences comprise or consist of a nucleotide sequence at least 90% identical to SEQ ID NO:

18.

21. The lentiviral vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a liver-specific promoter, preferably wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a hepatocyte-specific promoter.

22. The lentiviral vector of any of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a transthyretin (TTR) promoter, an alpha-1-antitrypsin (AAT) promoter, a thyroxine-binding globulin (TBG) promoter, an APoE / hAAT promoter, an HCR-hAAT promoter, an LP1 promoter, or an HLP promoter.

23. The lentiviral vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a transthyretin (TTR) promoter, preferably wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to an Enh1mTTR (ET) promoter.

24. The lentiviral vector of any one of the preceding claims, wherein the nucleotide sequence encoding the MMA-associated polypeptide is operably linked to a promoter comprising or consisting of: A nucleotide sequence that is at least 70% identical to SEQ ID NO:

19.

25. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is pseudotyped, preferably wherein the lentiviral vector is VSV.G pseudotyped.

26. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is a self-inactivating (SIN) lentiviral vector, preferably wherein the lentiviral vector comprises a self-inactivating (SIN) LTR, the LTR comprising or consisting of: A nucleotide sequence at least 70% identical to SEQ ID NO: 23, or a fragment thereof.

27. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector is an integrating lentiviral vector and / or a replication-defective lentiviral vector.

28. The lentiviral vector of any one of the preceding claims, wherein the lentiviral vector comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO:

36.

29. An isolated cell comprising the lentiviral vector according to any one of claims 1-28.

30. A pharmaceutical composition comprising a combination of the lentiviral vector according to any one of claims 1 to 28 and a pharmaceutically acceptable carrier, diluent or excipient.

31. The lentiviral vector according to any one of claims 1 to 28 or the pharmaceutical composition according to claim 30 for use as a medicament.

32. Use of the lentiviral vector according to any one of claims 1 to 28 or the pharmaceutical composition according to claim 30 in the preparation of a drug.

33. A method comprising administering a therapeutically effective amount of a lentiviral vector according to any one of claims 1-28 or a pharmaceutical composition according to claim 30 to a subject in need thereof.

34. The lentiviral vector according to any one of claims 1 to 28 or the pharmaceutical composition according to claim 30, for use in preventing or treating methylmalonic acidemia (MMA).

35. Use of the lentiviral vector according to any one of claims 1 to 28 or the pharmaceutical composition according to claim 30 in the preparation of a medicament for preventing or treating methylmalonic acidemia (MMA).

36. A method for preventing or treating methylmalonic acidemia (MMA), comprising administering a therapeutically effective amount of the lentiviral vector according to any one of claims 1 to 28 or the pharmaceutical composition according to claim 30 to a subject in need thereof.

37. The lentiviral vector or pharmaceutical composition for use according to claim 34, the use according to claim 35, or the method according to claim 36, wherein the MMA-associated polypeptide is MMUT or a fragment thereof, and the MMA is mut-type MMA (mut-MMA); wherein the MMA-associated polypeptide is MMAA or a fragment thereof, and the MMA is cblA-type MMA (cblA-MMA); wherein the MMA-associated polypeptide is MMAB or a fragment thereof, and the MMA is cblB-type MMA (cblB-MMA); wherein the MMA-associated polypeptide is MMADHC or a fragment thereof, and the MMA is cblD-type MMA (cblD-MMA); or wherein the MMA-associated polypeptide is MCEE or a fragment thereof, and the MMA is due to MCEE deficiency.

38. The lentiviral vector or pharmaceutical composition for use according to claim 34 or 37, the use according to claim 35 or 37, or the method according to claim 36 or 37, wherein the MMA-associated polypeptide is MMUT or a fragment thereof, and the MMA is mut-MMA, preferably wherein the mut-MMA is mut 0 Subtype or mut - Subtype.

39. The lentiviral vector or pharmaceutical composition for use according to any one of claims 31, 34, 37-38, the use according to any one of claims 32, 35, 37-38, or the method according to any one of claims 33, 36-39, wherein the subject is a human subject.

40. The lentiviral vector or pharmaceutical composition for use according to any one of claims 31, 34, 37-39, the use according to any one of claims 32, 35, 37-39, or the method according to any one of claims 33, 36-39, wherein the subject is a minor.

41. The lentiviral vector or pharmaceutical composition for use according to any one of claims 31, 34, 37-39, the use according to any one of claims 32, 35, 37-39, or the method according to any one of claims 33, 36-39, wherein the subject is a pediatric patient, preferably wherein the subject is a neonatal patient or an infant patient.

42. The lentiviral vector or pharmaceutical composition for use according to any one of claims 31, 34, 37-41, the use according to any one of claims 32, 35, 37-41, or the method according to any one of claims 33, 36-41, wherein the lentiviral vector or the pharmaceutical composition is administered systemically, preferably wherein the lentiviral vector or the pharmaceutical composition is administered by intravenous injection or intraperitoneal injection.

43. The lentiviral vector or pharmaceutical composition for use according to any one of claims 31, 34, 37-41, the use according to any one of claims 32, 35, 37-41, or the method according to any one of claims 33, 36-41, wherein the lentiviral vector or the pharmaceutical composition is administered locally, preferably wherein the lentiviral vector or the pharmaceutical composition is administered by direct injection, intra-arterial injection or intra-portal vein injection.

44. The lentiviral vector for use or the pharmaceutical composition according to claim 43, the use according to claim 43 or the method according to claim 43, wherein the lentiviral vector or the pharmaceutical composition is administered locally to the liver, preferably wherein the lentiviral vector or the pharmaceutical composition is administered by intrahepatic injection, intrahepatic arterial injection or intraportal vein injection.

45. The lentiviral vector or pharmaceutical composition for use according to any one of claims 31, 34, 37-44, the use according to any one of claims 32, 35, 37-43, or the method according to any one of claims 33, 36-44, wherein the lentiviral vector is expressed in at least about 10 8 TU / kg, at least about 10 9 TU / kg, or at least about 10 10 The dose of TU / kg was administered.

46. ​​The lentiviral vector or pharmaceutical composition for use according to any one of claims 31, 34, 37-45, the use according to any one of claims 32, 35, 37-45, or the method according to any one of claims 33, 36-45, wherein the lentiviral vector is expressed in about 10 8 to about 10 11 TU / kg, from about 10 8 to about 10 10 TU / kg, or about 10 9 to about 10 10 The dose of TU / kg was administered.

47. The lentiviral vector or pharmaceutical composition for use according to any one of claims 31, 34, 37-46, the use according to any one of claims 32, 35, 37-46, or the method according to any one of claims 33, 36-46, wherein the lentiviral vector is integrated into the genome of hepatocytes and maintained as a hepatocyte replica, preferably, wherein the lentiviral vector is integrated into the genome of hepatocytes and maintained as a hepatocyte replica.

48. The lentiviral vector or pharmaceutical composition for use according to any one of claims 34, 37-47, the use according to any one of claims 35, 37-47, or the method according to any one of claims 36-47, wherein the plasma methylmalonic acid level and / or the urine methylmalonic acid level is reduced and / or normalized.

49. The lentiviral vector or pharmaceutical composition for use according to any one of claims 34, 37-48, the use according to any one of claims 35, 37-48, or the method according to any one of claims 36-48, wherein mitochondrial function is improved, preferably wherein the plasma level of Fgf21 is reduced.

50. An immunoshielded lentiviral vector for use in a method of therapy, wherein the method comprises administering the immunoshielded lentiviral vector to a minor or pediatric subject.

51. A cell for use in a method of therapy, wherein the cell comprises an immune-shielded lentiviral vector, and wherein the method comprises administering the cell to a minor or pediatric subject.

52. An immunoshielded lentiviral vector or cell for use according to claim 50 or 51, wherein the subject is a neonatal subject or an infant subject.

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