Vector production

Abstract

Enveloped virus particle producer cells or packaging cells, wherein the cells are modified to reduce the expression of low-density lipoprotein receptors (LDLRs) on the cell surface.

Description

Technical Field

[0001] The present invention relates to cells and methods for producing enveloped virus particles. The present invention also relates to cells and methods for producing virus-like particles (VLPs). In particular, the present invention relates to cells modified to reduce the expression of low density lipoprotein receptor (LDLR) on the cell surface, and their use.

Background Art

[0002] Gene therapy involves the uptake of genetic material into cells to treat or prevent diseases. The genetic material can supplement defective genes with their functional copies, inactivate inappropriately functioning genes, or introduce new therapeutic genes into cells. Delivery of genetic material to cells may be achieved through the use of vectors that facilitate the transfer of nucleic acids. Viruses may be engineered to deliver a nucleotide of interest (NOI) to target cells and are commonly used as vectors in gene therapy. Viruses used in gene therapy to date include lentiviruses, retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), herpes simplex viruses (HSV), and vaccinia virus.

[0003] Lentiviral vectors (LVs), such as those derived from HIV-1, represent an efficient and versatile platform for gene therapy. LVs can deliver genes to both dividing and non-dividing cells, stably integrate into the genome of target cells, and possess relatively large cargo capacity. In addition, the prevalence of existing anti-vector immunization in humans is low. LVs are used in in vitro gene therapy and have shown promising results in preclinical studies, including direct in vivo administration such as liver-targeted gene therapy.

[0004] However, improvements in viral vector production are still greatly needed, for example, to obtain high yields and high-quality therapeutic products. One limiting factor may be the loss of infectious viral particles during the production process. For example, this may be at least partially due to reinfection of producer cells, which remain in contact with the produced virus (e.g., lentivirus) during production. This problem can become more severe when cells are grown in high-density suspension, a condition used in some cases for clinical-grade production. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The inventors have demonstrated that reducing the expression of low-density lipoprotein receptors (LDLRs) on the surface of cells used to produce enveloped virus particles leads to improved virus particle production.

[0006] LDLR is a transmembrane protein that recognizes the apolipoprotein that makes up LDL particles. When LDL particles bind to LDLR, the LDL particles are taken up into the cell and degraded while the LDLR is recycled to the cell surface. While we do not wish to be bound by theory, modifying cells to reduce LDLR expression on the surface could potentially disrupt the entry step during viral (e.g., lentivirus) infection.

[0007] The inventors increased the yield of lentivirus production by overexpressing proprotein convertase subtilisin kexin type 9 (PCSK9) to reduce the amount of LDLR on the surface of producer cells and consequently reduce reinfection during LV production. The inventors tested wild-type PCSK9 and the non-secretorized PCSK9.S127R mutant during LV production and evaluated the increase in titer and transduction ability of T cells both in vitro and in vivo in mice after intravenous (iv) administration.

[0008] The inventors demonstrated that the quantity of physical viral particles and their infectivity increased by using their modified cells. They further observed that the viruses produced using their modified cells contained increased levels of VSV-G on their surface. Furthermore, they showed that, surprisingly, the reduction in LDLR expression did not negatively affect the transduction efficiency of the produced viral particles, and even improved the transduction efficiency and vector copy number in transduced cells compared to the control.

[0009] The inventors' approach can be applied to enveloped virus-like particles (VLPs) and enveloped virus particles. [Means for solving the problem]

[0010] In one embodiment, the present invention provides enveloped virus particle producer cells or packaging cells that are modified to reduce the expression of low-density lipoprotein receptors (LDLRs) on the cell surface.

[0011] In another embodiment, the present invention provides enveloped virus particle packaging cells that are modified to reduce the expression of low-density lipoprotein receptors (LDLRs) on the cell surface.

[0012] Cells can be modified (e.g., genetically engineered) to overexpress, for example, the proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0013] In some embodiments, the cells contain heterogeneous polynucleotides that include a nucleotide sequence encoding the proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0014] In some embodiments, the expression of endogenous proprotein convertase subtilisin / kexin type 9 (PCSK9) is increased. For example, endogenous PCSK9 expression may be increased using a transcription activator (e.g., a manipulated transcription activator).

[0015] In another embodiment, the present invention relates to a method for producing enveloped virus particles, (a) A step of introducing a transfer vector and one or more helper vectors into cells, (b) A step of introducing a vector containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into cells, (c) A method is provided which includes the step of culturing cells under conditions suitable for the production of enveloped virus particles.

[0016] In some embodiments, steps (a), (b), and (c) are carried out sequentially in the order listed. In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, step (b) is performed before step (a) and optionally incorporates the nucleotide sequence encoding PCSK9 into the cell's genome.

[0017] This method can increase viral titer compared to the production of enveloped virus particles in the absence of heterologous polynucleotides or vectors containing the nucleotide sequence encoding PCSK9. This method can increase the physical quantity of enveloped virus particles compared to the production of enveloped virus particles in the absence of heterologous polynucleotides or vectors containing the nucleotide sequence encoding PCSK9.

[0018] The enveloped virus particles obtained by this method may exhibit increased transduction efficiency compared to enveloped virus particles produced in the absence of heterologous polynucleotides or vectors containing the nucleotide sequence encoding PCSK9. The enveloped virus particles obtained by this method may exhibit increased infectivity compared to enveloped virus particles produced in the absence of heterologous polynucleotides or vectors containing the nucleotide sequence encoding PCSK9.

[0019] In another embodiment, the present invention relates to a method for producing enveloped virus-like particles (VLPs), (a) A step of introducing a vector containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into enveloped virus particle packaging cells, (b) A method is provided which includes the step of culturing cells under conditions suitable for the production of enveloped VLPs.

[0020] In another embodiment, the present invention relates to a method for producing enveloped virus-like particles (VLPs), (a) A step of introducing one or more helper vectors into cells, (b) A step of introducing a vector containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into cells, (b) A method is provided which includes the step of culturing cells under conditions suitable for the production of enveloped VLPs.

[0021] In some embodiments, steps (a), (b), and (c) are carried out sequentially in the order listed. In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, step (b) is performed before step (a) and optionally incorporates the nucleotide sequence encoding PCSK9 into the cell's genome.

[0022] This method can increase VLP titer compared to the production of envelope VLPs in the absence of heterologous polynucleotides or vectors containing the nucleotide sequence encoding PCSK9. This method can increase the amount of physical VLPs compared to the production of envelope VLPs in the absence of heterologous polynucleotides or vectors containing the nucleotide sequence encoding PCSK9.

[0023] The enveloped VLPs obtained by this method may show increased transduction efficiency compared to enveloped VLPs produced in the absence of heterologous polynucleotides or vectors containing the nucleotide sequence encoding PCSK9. The enveloped VLPs obtained by this method may show increased infectivity compared to enveloped VLPs produced in the absence of heterologous polynucleotides or vectors containing the nucleotide sequence encoding PCSK9.

[0024] In another embodiment, the present invention provides enveloped virus particle producer cells or packaging cells, the cells comprising heterologous polynucleotides including a nucleotide sequence encoding a proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0025] In another embodiment, the present invention provides an enveloped virus particle packaging cell comprising heterologous polynucleotides containing a nucleotide sequence encoding a proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0026] In some embodiments, cells overexpress the proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0027] In some embodiments, PCSK9 is expressed transiently (e.g., from heterologous polynucleotides). Transient expression of PCSK9 may be less than about 96, 72, 48, or 24 hours, for example, about 12–72 hours, 12–48 hours, 12–24 hours, or 12–18 hours, for example, about 14–16 hours.

[0028] In some embodiments, PCSK9 is stably expressed (for example, from heterologous polynucleotides).

[0029] In some embodiments, enveloped virus particle producer cells or packaging cells are modified (e.g., genetically engineered) to constitutively express PCSK9.

[0030] In some embodiments, the heterologous polynucleotide is a vector. In some embodiments, the heterologous polynucleotide is a plasmid.

[0031] In some embodiments, heterologous polynucleotides are incorporated into the cell's genome.

[0032] In some embodiments, PCSK9 is human PCSK9.

[0033] In some embodiments, the nucleotide sequence encoding PCSK9 includes or comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding PCSK9 includes or comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 2.

[0034] In some embodiments, the nucleotide sequence encoding PCSK9 includes or consists of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding PCSK9 includes or consists of the nucleotide sequence of SEQ ID NO: 2.

[0035] In some embodiments, PCSK9 comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3. In some embodiments, PCSK9 comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 4.

[0036] In some embodiments, PCSK9 contains or consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, PCSK9 contains or consists of the amino acid sequence of SEQ ID NO: 4.

[0037] In a preferred embodiment, PCSK9 contains the mutation S127R, and the amino acids are numbered with reference to SEQ ID NO: 4.

[0038] In some embodiments, the cells are HEK-293 cells or derivatives thereof. In some embodiments, the cells are HEK-293T cells or HEK-293T-REx cells. In some embodiments, the cells are HEK-293T cells.

[0039] In some embodiments, the enveloped virus particles are or derived from lentiviruses, retroviruses, herpes simplex viruses, vaccinia viruses, hepadnaviruses, togaviruses, flaviviruses, arenaviruses, coronaviruses, orthomyxoviruses, paramyxoviruses, bunyaviruses, bornaviruses, rhabdoviruses, or filoviruses.

[0040] In a preferred embodiment, the enveloped virus particle is a lentivirus.

[0041] In a preferred embodiment, the enveloped virus particle is a pseudotype of vesicular stomatitis virus glycoprotein G (VSV-G). In a preferred embodiment, the enveloped virus particle is a VSV-G pseudotyped lentivirus.

[0042] In some embodiments, the lentivirus is a self-inactivating lentivirus.

[0043] In some embodiments, the enveloped VLP is a lentivirus VLP, retrovirus VLP, herpes simplex virus VLP, vaccinia virus VLP, hepadnavirus VLP, togavirus VLP, flavivirus VLP, arenavirus VLP, coronavirus VLP, orthomyxovirus VLP, paramyxovirus VLP, bunyavirus VLP, bornavirus VLP, rhabdovirus VLP, or filovirus VLP, or derived therefrom.

[0044] In a preferred embodiment, the envelope VLP is a lentivirus VLP.

[0045] In a preferred embodiment, the envelope VLP is a pseudotype of vesicular stomatitis virus glycoprotein G (VSV-G). In a preferred embodiment, the envelope VLP is a VSV-G pseudotype lentivirus VLP.

[0046] In some embodiments, the enveloped virus particle contains a polynucleotide comprising a transgene. In some embodiments, the transgene is a therapeutic transgene. In some embodiments, the transgene encodes a chimeric antigen receptor (CAR). In some embodiments, the transgene encodes Factor IX (FIX).

[0047] In some embodiments, the envelope VLP includes one or more components of a gene editing mechanism (e.g., CRISPR / Cas9, a base editor, or a prime editor).

[0048] In another aspect, the present invention provides a method for producing enveloped virus particle producer cells or packaging cells, wherein the cells are modified to reduce the expression of low-density lipoprotein receptor (LDLR) on the cell surface, and the method comprises introducing a polynucleotide containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into the cells. In another aspect, the present invention provides a method for producing enveloped virus particle packaging cells, wherein the cells are modified to reduce the expression of low-density lipoprotein receptor (LDLR) on the cell surface, and the method comprises introducing a polynucleotide containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into the cells. The introduction may be, for example, by transfusion or transduction. The introduction may be, for example, transient, or by integration of the nucleotide sequence encoding PCSK9 into the cell genome.

[0049] In another embodiment, the present invention provides the use of enveloped virus particle producer cells according to the present invention for the production of enveloped virus particles.

[0050] In another aspect, the present invention provides the use of the enveloped virus particle packaging cells of the present invention for the production of enveloped virus-like particles (VLPs).

[0051] In another embodiment, the present invention relates to a method for producing enveloped virus particles, (a) A step of providing an enveloped virus particle producer cell according to the present invention, (b) A method is provided comprising the step of culturing cells under conditions suitable for the production of enveloped virus particles.

[0052] In another embodiment, the present invention relates to a method for producing enveloped virus-like particles (VLPs), (a) A step of providing an enveloped virus particle packaging cell according to the present invention, (b) A method is provided which includes the step of culturing cells under conditions suitable for the production of enveloped VLPs.

[0053] In some embodiments, cells are cultured in a suspension. In some embodiments, the culturing is performed in a bioreactor.

[0054] In another embodiment, the present invention provides enveloped virus particles produced by the method of the present invention.

[0055] In another embodiment, the present invention provides enveloped virus-like particles (VLPs) produced by the method of the present invention.

[0056] In another aspect, the present invention provides cells transduced by the enveloped virus particles of the present invention.

[0057] In another aspect, the present invention provides cells transduced by enveloped virus-like particles (VLPs) of the present invention.

[0058] In some embodiments, the cells are T cells. In some embodiments, the cells are hematopoietic stem and / or progenitor cells (HSPCs).

[0059] In another embodiment, the present invention provides a pharmaceutical composition comprising enveloped virus particles or cells of the present invention and a pharmaceutically acceptable carrier, diluent, or excipient.

[0060] In another embodiment, the present invention provides a pharmaceutical composition comprising enveloped virus-like particles (VLPs) of the present invention and a pharmaceutically acceptable carrier, diluent, or excipient.

[0061] In another aspect, the present invention provides enveloped virus particles for therapeutic use. In another aspect, the present invention provides enveloped virus-like particles (VLPs) for therapeutic use. In another aspect, the present invention provides cells for therapeutic use.

[0062] In some embodiments, the treatment includes administering enveloped virus particles to a subject who requires them. In some embodiments, the treatment includes administering enveloped VLPs to a subject who requires them.

[0063] In another aspect, the present invention provides the use of enveloped virus particles of the present invention for the manufacture of a drug. In another aspect, the present invention provides the use of enveloped virus-like particles (VLPs) of the present invention for the manufacture of a drug. In another aspect, the present invention provides the use of cells of the present invention for the manufacture of a drug.

[0064] In another embodiment, the present invention provides a method of gene therapy comprising administering the enveloped virus particles of the present invention to a subject in need. In another embodiment, the present invention provides a method of gene therapy comprising administering enveloped virus-like particles (VLPs) of the present invention to a subject in need. In another embodiment, the present invention provides a method of gene therapy comprising administering the cells of the present invention to a subject in need.

[0065] In some embodiments, enveloped virus particles are administered systemically to the subject. In some embodiments, enveloped VLPs are administered systemically to the subject.

[0066] In some embodiments, enveloped virus particles are administered to subjects by intravenous injection. In some embodiments, enveloped VLPs are administered to subjects by intravenous injection. [Brief explanation of the drawing]

[0067] [Figure 1] This graph shows LV production accompanied by PCSK9 overexpression. A-C are the mean of infectivity titer (TU / mL, n=6, A), physical particle count (μg HIV Gag p24 / mL, n=6, B), or specific infectivity (TU / ng p24, n=6, C) with standard error of the mean (SEM) and single value for LV produced by adding pMAX.coPCSK9 (square) or pMAX.coPCSK9.S127R (triangle) to control PGK.GFP LV (circle), or translocation mixture. A pool of two independent experiments was used. D is the mean of PCSK9 concentration (μg / mL) in the LV-containing supernatant after LV production shown in (A-C), with SEM and single value, measured by ELISA. A pool of two independent experiments was used. E is a representative histogram of flow cytometry analysis of 293T cells, analyzed 54 hours after transfusion, either unstained (data drawn as gray lines) or stained with anti-LDLR antibody after the production of control PGK.GFP LV (data filled with gray lines) or LV produced by adding pMAX.coPCSK9.S127R to the transfusion mixture (data filled with black lines). [Figure 2]Graphs show the in vitro and in vivo evaluations of the transduction efficiency of LV produced by PCSK9 overexpression. A shows the mean vector copy number (VCN) measured 10 days after transduction in T cells transduced with control PGK.GFP LV (circles) or LV produced by adding pMAX.coPCSK9.S127R to the transduction mixture (triangles) at a multiplicity of infection (MOI) of 1 or 10 (n=2 for technical replication, n=3 for healthy donors). Graph B shows the mean human factor IX (hFIX) concentration in the plasma of mice treated with control ET.FIX LV (circles, n=10) or LV produced by adding pMAX.coPCSK9 (squares, n=5) or pMAX.coPCSK9.S127R (triangles, n=10) to the transduction mixture, with SEM. A pool of two independent experiments was used. C is the mean of VCN measured in the liver of mice shown in (B) 12 weeks after LV administration, with SEM and single values. A pool of two independent experiments was used. [Figure 3] This figure shows the quantitative determination of VSV.G content on the LV envelope. A and B are representative images obtained by immunostaining with anti-VSV.G antibody and analyzing by transmission electron microscopy LV(B) produced by adding control PGK.GFP LV(A) or pMAX.coPCSK9.S127R to a cleavage mixture. The scale bar is 200 nm. C is the average of the number of gold particles per virus particle immunostained with anti-VSV.G antibody, counted and analyzed by electron microscopy for control PGK.GFP LV (square, n=70), PGK.GFP LV produced using pMAX.coPCSK9.S127R (triangle, n=114), control ET.FIX LV (inverted triangle, n=83), ET.FIX LV produced using pMAX.coPCSK9.S127R (diamond, n=72), or ET.FIX LV produced without primary antibody (control, round, n=269), with SEM and single values. [Modes for carrying out the invention]

[0068] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including” or “includes,” or “containing” or “contains,” and are comprehensive or non-exclusive, not excluding additional unlisted components, elements, or processes. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0069] Low-density lipoprotein receptor (LDLR) In one embodiment, the present invention provides enveloped virus particle producer cells or packaging cells that are modified to reduce the expression of low-density lipoprotein receptors (LDLRs) on the cell surface.

[0070] The low-density lipoprotein (LDL) receptor (LDLR or LDL-R) is a protein that mediates the intracellular uptake of cholesterol-rich LDL. LDLR binds to LDL, the major cholesterol-carrying lipoprotein in plasma, and transports it into cells through intracellular uptake.

[0071] LDLR may be, for example, human LDLR.

[0072] An example of an LDLR amino acid sequence is as follows: MGPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISYKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDTEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLVFLCLGVFLLWKNWRLKNINSINFDNPVYQKTTEDEVHICHNQDGYSYPSRQMVSLEDDVA (SEQ ID NO: 5, Human LDLR)

[0073] In some embodiments, the LDLR comprises or consists of an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 5.

[0074] In some embodiments, PCSK9 comprises or consists of the amino acid sequence of SEQ ID NO: 5 or a fragment thereof.

[0075] LDLR fragments and / or variants may retain LDLR activity (e.g., the activity of SEQ ID NO: 5). For example, LDLR fragments and / or variants may bind to LDL and transport it into the cell by intracellular uptake. Preferably, LDLR fragments and / or variants may have the same or similar activity as LDLR, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of LDLR (e.g., LDLR of SEQ ID NO: 5). Those skilled in the art will be able to generate fragments and / or variants based on the known structural and functional characteristics of LDLR, for example, by using conservative substitutions.

[0076] A decrease in LDLR expression on the cell surface refers to a decrease in the number of LDLR molecules expressed on the surface of modified (e.g., genetically engineered) cells compared to the number of LDLR molecules expressed on the surface of cells under otherwise substantially identical conditions, but without modification.

[0077] The expression of LDLR on the cell surface can be reduced such that the number of surface-exposed LDLR molecules is less than, for example, 75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the number of surface-exposed LDLR molecules presented in the absence of the modification. The expression of LDLR on the cell surface can be readily determined by those skilled in the art using any suitable method known in the art, such as flow cytometry (as described herein).

[0078] In addition to decreased LDLR expression, the expression of one or more other LDLR family members may also be decreased. LDLR family members may include the very low-density lipoprotein receptor (VLDL receptor, VLDLR), ApoER2 (also known as LRP8), low-density lipoprotein receptor-related protein 4 (also known as multiple epidermal growth factor (EGF) repeat-containing protein (MEGF7)), LDLR-related protein 1, LDLR-related protein 1b, and megalin.

[0079] A decrease in the expression of LDLR family members on the cell surface refers to a decrease in the number of LDLR family member molecules expressed on the surface of a modified (e.g., genetically engineered) cell compared to the number of LDLR family member molecules expressed on the surface of a cell that is otherwise substantially identical, but lacks modification.

[0080] The expression of LDLR family members on the cell surface can be reduced such that the number of surface-exposed LDLR family member molecules is less than, for example, 75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the number of surface-exposed LDLR family member molecules presented in the absence of modification. The expression of LDLR family members on the cell surface can be readily determined by those skilled in the art using any suitable method known in the art, such as flow cytometry.

[0081] Proprotein convertase subtilisin kexin type 9 (PCSK9) Producer cells or packaging cells may be modified to overexpress, for example, proprotein convertase subtilisin / kexin type 9 (PCSK9). In some embodiments, the cells contain heterologous polynucleotides including a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0082] An example of the PCSK9 amino acid sequence is as follows: MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQP PDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPV TLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEG VYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ (Sequence No. 4)

[0083] In some embodiments, PCSK9 comprises or consists of an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 4.

[0084] In some embodiments, PCSK9 comprises or consists of the amino acid sequence of SEQ ID NO: 4 or a fragment thereof.

[0085] In a preferred embodiment, PCSK9 contains the mutation S127R, and the amino acids are numbered with reference to SEQ ID NO: 4.

[0086] The terms "corresponding to," "reference to," and "relative to," when used in the context of numbering a given amino acid or polynucleotide sequence, can refer to the numbering of residues in a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified relative to the reference sequence, not to the actual numerical positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of PCSK9, can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of residues in the given amino acid or polynucleotide sequence is done relative to the reference sequence to which it is aligned.

[0087] An example of the PCSK9 S127R amino acid sequence is as follows: MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMRGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQP PDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPV TLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEG VYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ (Sequence ID 3)

[0088] In some embodiments, PCSK9 comprises or consists of an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3. Preferably, PCSK9 comprises mutation S127R, and the amino acids are numbered with reference to SEQ ID NO: 4.

[0089] In some embodiments, PCSK9 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or a fragment thereof.

[0090] Examples of nucleotide sequences encoding PCSK9 include the following: (Sequence ID 2, Codon-optimized PCSK9) (Sequence ID 1, Codon-optimized PCSK9 S127R)

[0091] In some embodiments, the nucleotide sequence encoding PCSK9 comprises or consists of a nucleotide sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 2.

[0092] In some embodiments, the nucleotide sequence encoding PCSK9 includes or consists of the nucleotide sequence of SEQ ID NO: 2 or a fragment thereof.

[0093] In some embodiments, the nucleotide sequence encoding PCSK9 comprises or consists of a nucleotide sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 1. Preferably, PCSK9 comprises the mutation S127R, and the amino acids are numbered with reference to SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding PCSK9 comprises or consists of the nucleotide sequence or fragment thereof of SEQ ID NO: 1.

[0094] PCSK9 fragments and / or variants may retain PCSK9 activity (e.g., the activity of SEQ ID NO: 4 or 3). For example, PCSK9 fragments and / or variants may act as negative regulators of LDLR, enhancing its degradation upon binding. Preferably, PCSK9 fragments and / or variants may have the same or similar activity as PCSK9, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of PCSK9 (e.g., PCSK9 of SEQ ID NO: 4 or 3). Those skilled in the art will be able to generate fragments and / or variants based on the known structural and functional characteristics of PCSK9, for example, by using conservative substitutions.

[0095] The "heterogeneous" polynucleotide (e.g., a heterogeneous polynucleotide containing a nucleotide sequence encoding PCSK9) may be a polynucleotide that does not naturally exist in cells and is introduced into cells by any preferred method, such as transduction or transtransfer. The heterogeneous polynucleotide may be a vector, such as an expression vector. The heterogeneous polynucleotide may be a plasmid, such as a plasmid.

[0096] In some embodiments, the nucleotide sequence encoding PCSK9 is operably ligated to a promoter. The promoter may be, for example, a CMV promoter.

[0097] Examples of promoter sequences are as follows: GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA ATGACGTATGTTCCCATAGTAACGCCAATGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATG GCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGAT TTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGGT (Sequence ID 30)

[0098] In some embodiments, the nucleotide sequence encoding PCSK9 is operably linked to a polyadenylation signal. The polyadenylation signal may be, for example, an SV40 polyadenylation signal.

[0099] Examples of polyadenylation signals are as follows: GCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT (Sequence ID 31)

[0100] In some embodiments, the nucleotide sequence encoding PCSK9 is operably ligated to a chimeric intron.

[0101] Examples of chimeric intron sequences are as follows: GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGGCCATAGAAACTGGGCTTGTCGAGACAGAGAAGATTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG (Sequence ID 32)

[0102] Method of production In one embodiment, the present invention provides a method for producing enveloped virus particles (e.g., lentiviral particles).

[0103] The production method comprises (a) introducing a transfer vector and optionally one or more helper vectors into a cell (e.g., a host cell), and (b) culturing the cell under conditions suitable for producing enveloped virus particles (e.g., lentiviral particles) according to the present invention. Enveloped virus particles (e.g., lentiviral particles) may then be obtained from the cell. As used herein, “transfer vector” may encode the genome of the virus (e.g., lentivirus) of the present invention. Preferably, the transfer vector used to produce a lentiviral genome in a host cell / packaging cell will have sufficient lentiviral genetic information to enable the packaging of the RNA genome into viral particles that can infect target cells in the presence of packaging components (e.g., gag-pol, rev, env) but cannot independently replicate to produce infectious viral particles in the final target cell.

[0104] Transfer vectors used to produce a viral genome within host cells / packaging cells may contain transcriptional regulatory sequences operably ligated to the lentiviral genome to direct the transcription of the genome within the cell. These regulatory sequences may be native sequences related to the transcribed viral sequence (i.e., the 5' U3 region), or they may be heterologous promoters, such as another viral promoter (e.g., the CMV promoter). The transfer vector may also be a plasmid.

[0105] As used herein, a “helper vector” may encode one or more packaging components (e.g., gag-pol, rev, env). The nucleotide sequences encoding the packaging component(s) may be operably ligated to a promoter (e.g., a CMV promoter or an RSV promoter) and / or a polyadenylation signal. The term “helper vector” may 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 helper vector, packaging vector, and / or envelope vector may be plasmids.

[0106] In another embodiment, the present invention provides a method for producing enveloped virus-like particles (VLPs) (e.g., lentiviral VLPs).

[0107] In another aspect, the present invention provides a method for producing enveloped virus-like particles (VLPs), comprising: (a) introducing one or more helper vectors into cells; (b) introducing a vector containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into cells; and (b) culturing the cells under conditions suitable for the production of enveloped VLPs.

[0108] A transfer vector and / or one or more helper vectors can be introduced into host cells by any suitable technique known in the art, such as translocation, transduction, and / or transformation. Preferably, the helper vector may be transiently translocated or transduced into host cells, or stably maintained within host cells (e.g., stably integrated into the cellular genome). Alternatively, a combination of transient translocation or transduction and stable maintenance may be used to introduce the helper vector into host cells.

[0109] Preferably, the transfer vector and / or helper vector may be plasmids that can be introduced by transduction. Preferably, a four-plasmid system consisting of one transfer plasmid and three helper plasmids may be used. The three helper plasmids may consist of a first helper plasmid encoding the gag-pol gene, a second helper plasmid encoding the rev gene, and a third helper plasmid encoding the env gene. Alternatively, a three-plasmid system consisting of one transfer plasmid, one helper plasmid encoding the gag-pol and rev genes, and one helper plasmid encoding the env gene may be used. Alternatively, a two-plasmid system may be used in which all helper functions (e.g., gag-pol, rev, and env) are encoded by a single helper plasmid.

[0110] Enveloped virus particles (e.g., lentiviral particles) can be produced using any suitable host cell. Enveloped VLPs (e.g., lentiviral VLPs) can be produced using any suitable host cell. Suitable cells include producer cells and packaging cells, such as those described herein (e.g., HEK293 or its derivatives). Suitable conditions for culturing cells are well known to those skilled in the art. For example, cells may be cultured in culture medium (e.g., chemically defined medium) for about 1 to about 5 days (e.g., about 48 hours, about 54 hours, or about 72 hours). For example, cells may be cultured in culture medium (e.g., chemically defined medium) for about 2 to about 3 days.

[0111] Enveloped virus particles (e.g., lentiviral particles) may be obtained using any suitable method known in the art. For example, the culture supernatant may be collected and then the enveloped virus particles (e.g., lentiviral particles) may be purified from the culture supernatant (e.g., by centrifugation, membrane filtration and / or chromatography). The production method may further include any other suitable process steps, such as DNA reduction, concentration, formulation and / or sterilization.

[0112] Enveloped VLPs (e.g., lentivirus VLPs) may be obtained using any suitable method known in the art. For example, the culture supernatant may be collected, and then the enveloped VLPs (e.g., lentivirus VLPs) may be purified from the culture supernatant (e.g., by centrifugation, membrane filtration and / or chromatography). The production method may further include any other suitable process steps, such as DNA reduction, concentration, formulation and / or sterilization.

[0113] Kits and Systems In one embodiment, the present invention provides a kit or system for producing enveloped virus particles (e.g., lentiviral particles) according to the present invention.

[0114] The kit or system may include an enveloped virus particle (e.g., lentiviral particle) producer cell or packaging cell of the present invention. The kit or system may further include a transfer vector encoding the lentiviral genome of the present invention and optionally one or more helper vectors. The kit or system may further include other reagents (e.g., translocation reagents, culture media, etc.). The kit or system may further include any other suitable components and optionally instructions for producing the enveloped virus particle (e.g., lentiviral particle) of the present invention.

[0115] In one embodiment, the present invention provides a kit or system for producing the envelope VLP (e.g., lentivirus VLP) of the present invention.

[0116] The kit or system may comprise the enveloped virus particle packaging cells of the present invention. The kit or system may further comprise one or more helper vectors. The kit or system may further comprise other reagents (e.g., translocation reagents, culture media, etc.). The kit or system may further comprise any other suitable components and, optionally, instructions for producing the enveloped VLPs (e.g., lentiviral VLPs) of the present invention.

[0117] cell The cells may be isolated cells. Preferably, the cells are mammalian cells, such as human cells. The cells may be isolated human cells.

[0118] Preferably, the cells may be producer cells. The term “producer cells” can refer to cells that produce viral particles, for example, that are transiently transfected, stably transfected, and / or transduced with all the elements necessary to produce viral particles. Suitable producer cells are well known to those skilled in the art and may include HEK293, COS-1, COS-7, CV-1, HeLa, CHO, and A549 cell lines. In some embodiments, the producer cells are HEK293 cells or their derivatives (e.g., HEK293T cells, HEK293T Lenti-X, HEK293T-Rex cells, HEK293FT cells, HEK293SF-3F6 cells, HEK293SF-3F9 cells, HEK293-EBNA1 cells, or SJ293TS cells).

[0119] Preferably, the cells may be packaging cells. The term “packaging cells” may refer to cells that contain some or all of the elements necessary for packaging a recombinant viral genome. 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 are incorporated into the genome of the packaging cell. Cells containing only some of the elements necessary for the production of enveloped viral particles are useful as intermediate reagents in the generation of viral particle producer cell lines through subsequent steps of transient translocation, transduction, or stable incorporation of each additionally required element. These intermediate reagents are encompassed under the term “packaging cells.”

[0120] Packaging cells lacking transfer vectors do not contain sequences encoding the viral genome and can therefore be used for the production of enveloped VLPs.

[0121] Enveloped virus particles A vector is a tool that enables or facilitates the movement of an entity from one environment to another. The virus particles of the present invention may be vectors.

[0122] The viral vector particles of the present invention are enveloped virus particles.

[0123] Enveloped virus particles contain an outer lipid bilayer. Numerous enveloped viruses are known in the art, including lentiviruses, retroviruses, herpes simplex viruses, vaccinia viruses, hepadnaviruses, togaviruses, flaviviruses, arenaviruses, coronaviruses, orthomyxoviruses, paramyxoviruses, bunyaviruses, bornaviruses, rhabdoviruses, and filoviruses.

[0124] The enveloped virus particle producer cells or packaging cells of the present invention and the method for producing enveloped virus particles of the present invention may be for the production of enveloped virus particles (preferably lentiviral particles) as disclosed herein.

[0125] lentiviral vectors Lentiviral vectors may be in the form of lentiviral particles. In some embodiments, lentiviral vectors may include a lentiviral genome. As used herein, a lentiviral genome may refer to a genome containing at least one element that is derived from or derivable from a lentiviral genome. Lentiviruses are a genus of retroviruses, which contain an RNA genome that is converted to DNA by reverse transcriptase in transduced cells. Lentiviral vectors can transduce a wide range of cell types, incorporate the host genome in both dividing and postmittal cells, and result in long-term expression of protein-coding sequences both in vitro and in vivo.

[0126] The basic genes necessary for the survival and function of lentiviruses are the gag, pol, and env genes, where gag encodes a structural protein, pol encodes an enzyme necessary for reverse transcription and integration into the host cell genome, and env encodes a viral envelope glycoprotein. Lentiviruses may also have additional cis-acting elements, such as a rev response element (RRE) that enables efficient transport of the integrated proviral RNA transcript from the nucleus to the cytoplasm of infected target cells, a retroviral psi packaging element involved in regulating the essential process of packaging the retroviral RNA genome into the viral capsid during replication, a primer binding site (PBS) where reverse transcription is initiated, a TAT activation region (TAR), splice donor and splice acceptor sites, and central and terminal polyprint lactates that enable the initiation of positive-strand synthesis.

[0127] In lentiviral genomes, elements are typically flanked at both ends by regions called long-terminal repeats (LTRs). LTRs are responsible for integration and transcription. LTRs may also function as enhancer-promoter sequences, regulating the expression of lentiviral genes. LTRs themselves are identical or nearly identical sequences and can typically be divided into three regions: U3, R, and U5. LTRs may be naturally occurring or modified. For example, U3 and U5 modifications are described in Iwakuma et al. (1999) Virology 261:120-132.

[0128] The lentiviral particles of the present invention may include a minimal lentiviral genome. As used herein, a minimal lentiviral genome may mean that the lentiviral genome has been manipulated to remove non-essential elements and retain essential elements in order to provide the functionality necessary to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell (see, for example, Kim et al. (1998) Journal of Virology 72:811-816; Sertkaya et al. (2021) Scientific Reports 11:1-15).

[0129] A lentiviral genome may include, from 5' to 3', a 5'LTR, one or more lentiviral cis-acting elements, and a 3'LTR. Preferably, a lentiviral genome may include, from 5' to 3', a 5'LTR, an RRE, and a 3'LTR. Preferably, a lentiviral genome may include, from 5' to 3', a 5'LTR, a retroviral psi packaging element, an RRE, and a 3'LTR. Preferably, a lentiviral genome may include, from 5' to 3', a 5'LTR, a retroviral psi packaging element, an RRE, a cPPT, and a 3'LTR. Preferably, a lentiviral genome may include, from 5' to 3', a 5'LTR, a PBS, a retroviral psi packaging element, an RRE, a cPPT, and a 3'LTR.

[0130] A lentiviral genome may further include a protein-coding sequence and, optionally, one or more regulatory elements (e.g., operably linked to the protein-coding sequence). Preferably, a lentiviral genome may include, from 5' to 3', a 5'LTR, an RRE, a protein-coding sequence, and a 3'LTR. Preferably, a lentiviral genome may include, from 5' to 3', a 5'LTR, a retroviral psi packaging element, an RRE, a protein-coding sequence, and a 3'LTR. Preferably, a lentiviral genome may include, from 5' to 3', a 5'LTR, a retroviral psi packaging element, an RRE, a cPPT, a protein-coding sequence, and a 3'LTR. Preferably, a lentiviral genome may include, from 5' to 3', a 5'LTR, a PBS, a retroviral psi packaging element, an RRE, a cPPT, a protein-coding sequence, and a 3'LTR.

[0131] The lentiviral particles 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 lentivirus "replication-deficient" or "non-replicational." Preferably, one or more of the gag, pol, rev, and env genes are (at least partially) deleted in the replication-deficient lentivirus. Preferably, each of the gag, pol, rev, and env genes is (at least partially) deleted in the replication-deficient lentivirus. Optionally, the lentivirus lacks the functional gag-pol and / or env genes and / or other genes essential for replication.

[0132] The lentiviral particles of the present invention may be derived from any lentivirus. As used herein, “lentiviral-derived” or “lentiviral-based” may mean that the lentiviral genome contains one or more elements from the lentivirus. For example, the coding region of a viral protein may be deleted, but one or more cis-acting elements may be retained from the lentivirus. The lentiviral particles may be derived from primate lentiviruses. Examples of “primate” lentiviruses include, but are not limited to, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The lentiviral particles may be derived from non-primate lentiviruses (i.e., lentiviruses that do not primarily infect primates, particularly humans). Examples of "non-primate" lentiviruses include, but are not limited to, the prototype "slow virus" visna / maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious aneemia virus (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[0133] Preferably, the lentiviral particles of the present invention are HIV-derived lentiviral particles. As used herein, “HIV-derived” or “HIV-based” may mean that the lentiviral genome contains one or more elements from HIV. For example, the coding region of an HIV viral protein may be deleted, and one or more HIV cis-active elements may be retained in the lentiviral genome (see, for example, Johnson (2021) Molecular Therapy-Methods & Clinical Development 21:451-465). An HIV-derived lentiviral genome may include, from 5' to 3', a 5'LTR, one or more HIV-derived cis-active elements (e.g., RRE and / or cPPT), and a 3'LTR.

[0134] Preferably, the lentiviral particles of the present invention are HIV-1-derived lentiviral particles. The prototype lentiviral vector system is based on HIV-1 (see, for example, Merten et al. (2016) Molecular Therapy-Methods & Clinical Development 3:16017). The sequence extending into the gag open reading frame has been shown to be important for the packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of the gag with a mutated translation start codon. In addition, HIV-1 vectors also often contain a portion of the env gene containing the RRE. The rev binds to the RRE, which allows for the transport of full-length or single-spliced ​​mRNA from the nucleus to the cytoplasm. In the absence of the rev and / or RRE, full-length HIV-1 RNA can accumulate in the nucleus. Alternatively, the need for the rev and RRE can be reduced by using constitutive transport elements from certain simple retroviruses, such as the Mason-Pfizer monkey virus. An HIV-1 lentiviral genome may include, from 5' to 3', a 5'LTR, one or more HIV-1-derived cis-acting elements (e.g., PBS, retroviral psi packaging element, RRE, and / or cPPT), and a 3'LTR.

[0135] The lentiviral particles of the present invention may also be self-inactivated lentiviral particles. As used herein, “self-inactivated” lentiviral particles or “SIN” lentiviral particles may contain a lentiviral genome in which the lentiviral enhancer sequence and the lentiviral promoter sequence are deleted (see, for example, Zufferey (1998) Journal of Virology 72:9873-9880; Miyoshi et al. (1998) Journal of Virology 72:8150-8157). SIN lentiviral particles can generate and transduce non-dividing cells in vivo with similar efficacy to wild-type vectors. Transcriptional inactivation of the long-terminal repeat (LTR) in the SIN provirus can prevent recruitment by a replicable virus. This can also enable regulated expression of genes from the internal promoter by eliminating any cis-acting effects of the LTR.

[0136] The lentiviral vector particles of the present invention may be integration-capable. As used herein, “integration-capable” lentiviral vectors can be integrated into the genome of a host cell. In contrast to integration-capable lentiviral vectors, integration-defective lentiviral vectors (IDLVs) can be produced, for example, by packaging the lentiviral vector with a catalytically inactive integrase (such as HIV integrase having 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, for example, Wanisch et al. (2009) Molecular Therapy 17:1316-1332).

[0137] The lentiviral particles of the present invention may be replication-defective and incorporated. The lentiviral particles of the present invention may be replication-defective, incorporated, and self-inactivated. The lentiviral particles of the present invention may be replication-defective, incorporated, self-inactivated, and HIV-derived.

[0138] "Lentiviral particles" may refer to a lentiviral genome having an envelope. Lentiviral particles can be produced by the simultaneous translocation into a host cell of a plasmid containing the lentiviral genome (e.g., a "transfer vector") and a helper plasmid (e.g., a "packaging vector" encoding gag-pol and / or rev, and an "envelope vector" encoding env), followed by the recovery of the lentiviral supernatant.

[0139] The lentiviral particles of the present invention may be pseudotyped. Pseudotyped lentiviral particles having a naturally occurring or engineered lentiviral envelope may enable targeted transduction of specific cell types (see, for example, Joglekar et al. (2017) Human Gene Therapy Methods 28:291-301).

[0140] Preferably, the lentiviral particles of the present invention are VSV-G pseudotypes. Vesicular stomatitis virus G protein (VSV-G) is an envelope protein commonly used for pseudotype formation. VSV-G is a trimer protein that binds to phosphatidylserine and low-density lipoprotein receptors on the cell surface and invaginates into the cell plasma membrane.

[0141] The lentiviral particles of the present invention may be replication-deficient, integrated, and VSV-G pseudotyped. The lentiviral particles of the present invention may be replication-deficient, integrated, self-inactivated, and VSV-G pseudotyped. The lentiviral particles of the present invention may be replication-deficient, integrated, self-inactivated, HIV-derived, and VSV-G pseudotyped.

[0142] In some embodiments, the lentiviral particles of the present invention (i) contain one or more miRNA target sequences, (ii) are CD47 high lentiviral particles, and / or (iii) are MHC-I free lentiviral particles. In preferred embodiments, the lentiviral particles of the present invention contain one or more miRNA target sequences and are CD47 high lentiviral particles / MHC-I free lentiviral particles. Each of these features may reduce the immune response after administration.

[0143] In some embodiments, the envelope virus particle producer cells or packaging cells of the present invention are CD47 high producer cells or packaging cells. In some embodiments, the envelope virus particle producer cells or packaging cells of the present invention are MHC-I low producer cells or packaging cells. In some embodiments, the envelope virus particle producer cells or packaging cells of the present invention are MHC-I free producer cells or packaging cells.

[0144] The envelope virus particle producer cells or packaging cells of the present invention are CD47 high / MHC-I free producer cells or packaging cells or CD47 high / MHC-I low may be producer cells or packaging cells. In preferred embodiments, the envelope virus particle producer cells or packaging cells of the present invention are CD47 high / MHC-I free producer cells or packaging cells.

[0145] miRNA target sequence The lentiviral particles of the present invention may contain one or more miRNA target sequences. The one or more miRNA target sequences may be operably ligated to a protein-coding sequence. The term "operably ligated" may mean that the described components are in a relationship that allows them to function in their intended manner.

[0146] MicroRNA (miRNA) genes are scattered across all human chromosomes except the Y chromosome. Similar to protein-coding genes, miRNAs are typically transcribed from the polymerase II promoter, producing a primary miRNA transcript (pri-miRNA). From the pri-miRNA, a stem-loop approximately 60 nucleotides long, called a pre-miRNA, is cleaved, leaving a 5' phosphate group and a 2 bp-long 3' overhang. The pre-miRNA is then actively transported from the nucleus to the cytoplasm. Dicer then performs a double-strand break at the other end of the stem-loop, resulting in mature miRNA and miRNA. * It generates a 19-24 bp double helix, consisting of a double helix called a pycnocopter and its opposite strand. One strand of the double helix is ​​selectively loaded into the RNA-induced silencing complex (RISC) and accumulates as a mature microRNA. This strand is usually the one whose 5' end pairs more weakly to its complement. However, there are some miRNAs that support the accumulation of both strands of the double helix to an equal degree.

[0147] When loaded into RISC, the guide strand of a mature microRNA interacts with the mRNA target sequence, which is preferentially found in the 3' untranslated region (3'UTR) of the protein-coding gene. If the entire guide strand sequence is perfectly complementary to the mRNA target, the mRNA is cleaved in a endonuclear degradation manner. If only the seed sequence (i.e., nucleotides 2-8 from the 5' end of the miRNA) is perfectly complementary to the target mRNA, RNAi may act via an alternative mechanism that results in translational repression.

[0148] Protein expression from a protein-coding sequence (i.e., "transgene expression") may be regulated by one or more endogenous miRNAs using one or more corresponding miRNA target sequences. Using this method, one or more miRNAs endogenously expressed in a cell prevent or reduce transgene expression in that cell by interacting with their corresponding miRNA target sequences located in the lentiviral genome.

[0149] Suitable miRNA target sequences for suppressing transgene expression in specific cells are known to those skilled in the art. Determining miRNAs with a desired expression profile may be achieved using techniques known to those skilled in the art. For example, a mammalian microRNA expression atlas is described in Landgraf et al. (2007) Cell 129:1401-1414, and the distribution of miRNA expression across human tissues is described in Ludwig et al. (2016) Nucleic Acids Research 44:3865-3877. After a miRNA is identified, the corresponding target sequence can be easily determined using a microRNA database such as miRBase (Griffiths-Jones et al. (2007) Nucleic Acids Research 36(suppl_1):D154-D158).

[0150] A miRNA target sequence may be fully or partially complementary to the corresponding miRNA. As used herein, the term “fully complementary” may mean that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it. As used herein, the term “partially complementary” may mean that the target sequence is only partially complementary to the sequence of the miRNA that recognizes it, thereby the partially complementary sequence is still recognized by the miRNA. In other words, a partially complementary target sequence in the context of the present invention is effective in recognizing the corresponding miRNA and resulting in the prevention or reduction of transgene expression in cells expressing that miRNA. Preferably, a partially complementary miRNA target sequence may be fully complementary to the miRNA seed sequence.

[0151] Including two or more copies of a miRNA target sequence in a lentiviral vector can increase the effectiveness of the system. It may also include different miRNA target sequences. For example, a protein-coding sequence may be operably ligated to multiple miRNA target sequences, which may or may not be different. The miRNA target sequences may be in series, but other arrangements are also conceivable. A lentiviral vector may contain, for example, one, two, three, four, five, six, seven, or eight copies of the same or different miRNA target sequences. Preferably, the lentiviral vector contains four of the miRNA target sequences from each miRNA target sequence.

[0152] Copies of the miRNA target sequence may be separated by a spacer sequence. The spacer sequence may, for example, contain at least one, at least two, at least three, at least four, or at least five nucleotide bases.

[0153] Preferably, 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. Preferably, the protein coding sequence is operably ligated 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 ligated to four miRNA target sequences.

[0154] The miRNA target sequence may be a human miRNA target sequence. Preferably, the miRNA target sequence is a -5p or -3p miRNA target sequence.

[0155] One or more miRNA target sequences may suppress transgene expression in one or more cells other than hepatocytes (e.g., hepatocytes).

[0156] One or more miRNA target sequences may suppress transgene expression in hematopoietic cells. Hematopoietic stem cells give rise to different types of blood cells in lineages called the myeloid and lymphoid lineages. As used herein, “hematopoietic cells” may include myeloid cells and lymphoid cells. Myeloid cells may include monocytes, macrophages, neutrophils, basophils, and eosinophils. Lymphoid cells may include T cells, B cells, natural killer cells, and innate lymphoid cells.

[0157] One or more miRNA target sequences can suppress transgene expression in antigen-presenting cells. As used herein, “antigen-presenting cell” (APC) may refer to a cell that presents an antigen bound by major histocompatibility complex (MHC) proteins on its surface. APCs may be hematopoietic cells. Antigen-presenting cells may be professional antigen-presenting cells. Professional APCs are specialized in presenting antigens to T cells and may include macrophages, B cells, and dendritic cells. Preferably, APCs are splenic APCs and / or hepatic APCs.

[0158] One or more miRNA target sequences can suppress transgene expression in hematopoietic antigen-presenting cells.

[0159] By preventing transgene expression in antigen-presenting cells while allowing high levels of expression in other cells, miRNA regulation can enable robust and stable gene transfer in the absence of an immune response.

[0160] As used herein, the term “suppress expression” may mean a reduction in the expression of a transgene in a relevant cell type (maybe one) to which one or more miRNA target sequences are operably ligated, compared to transgene expression under otherwise substantially identical conditions, but in the absence of one or more miRNA target sequences. In some embodiments, transgene expression is suppressed by at least 50%. In some embodiments, transgene expression is suppressed by at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, transgene expression is substantially inhibited, for example, undetectable.

[0161] miRNA-mediated approaches to restrict gene expression offer several advantages over other strategies for modulating transgenes. While tissue-specific promoters can successfully restrict expression in target cells, leaky expression in non-target cell fractions is observed. This is because reconstituted promoters, modified for inclusion in vector systems, often lose some of their cell specificity, and also because vector incorporation near active promoters and enhancers can activate tissue-specific promoters, driving transgene expression. In contrast, miRNA-mediated silencing occurs at the post-transcriptional level, making promoter-trapping and enhancer-trapping irrelevant. Therefore, miRNA regulation can be used to effectively detarget transgene expression from specific cell types while still allowing broad tissue expression. miRNA regulation can also be used in combination with tissue-specific promoters / enhancers. An additional regulatory layer is added that can eliminate off-target expression by including miRNA target sequences in an expression cassette already under the control of a tissue-specific promoter.

[0162] Exemplary miRNA target sequences that suppress transgene expression in hematopoietic cells and / or antigen-presenting cells include, but are not limited to, the miR-142, miR-181, miR-223, and miR-155 target sequences. Other miRNA target sequences that suppress transgene expression in hematopoietic cells and / or antigen-presenting cells are known in the art (see, for example, Ghafouri-Fard et al. (2021) Non-coding RNA Research 6:8-14). miRNAs expressed in hematopoietic cells and / or antigen-presenting cells interact with their corresponding miRNA target sequences to reduce the expression of target genes.

[0163] Further miRNA target sequences that suppress transgene expression in hematopoietic cells and / or antigen-presenting cells can be identified by any suitable method, e.g., Monticelli et al. (2005) Genome Biology 6:1-15.

[0164] CD47 high lentivirus particles The lentiviral particles of the present invention are CD47 high Lentiviral particles may also be used. When used herein, "CD47" high A "lentiviral particle" may refer to a lentiviral particle having an increased level of CD47 (or fragments thereof) on its surface. high Lentiviral particles may have reduced uptake by professional phagocytic cells. In some embodiments, CD47 high The surface of the lentiviral particles contains higher levels of CD47 protein than control lentiviral particles (AT CC® CRL-11268®) produced in HEK293T cells.

[0165] CD47 (surface antigen classification 47), also known as integrin-associated protein (IAP), is a transmembrane protein encoded by the CD47 gene in humans. Phagocytosis is physiologically inhibited by CD47, a ligand for the ubiquitously expressed signal regulatory protein α (SIRP-α) receptor, which is expressed by professional phagocytic cells. CD47 can be incorporated into lentiviral particles when they emerge from producer cells as axillary buds.

[0166] The lentiviral particles of the present invention may contain 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 phagocytic cells. Any suitable analytical method may be used to quantify the amount of CD47 polypeptide (or fragments thereof) present on the surface of the lentiviral particles.

[0167] In some embodiments, the density of the CD47 polypeptide (or fragment thereof) can be determined by immunostaining and total internal reflection fluorescence microscopy of CD47, for example, as described in U.S. Patent Application Publication No. 20100316570(A1). The CD47 polypeptide (or fragment thereof) is at least about 20 molecules / μm 2 at least approximately 25 molecules / μm 2 at least approximately 30 molecules / μm 2 at least approximately 35 molecules / μm 2 at least about 40 molecules / μm 2 , at least about 45 molecules / μm 2 at least approximately 50 molecules / μm 2 at least about 60 molecules / μm 2 at least approximately 70 molecules / μm 2 , at least about 80 molecules / μm 2 at least approximately 90 molecules / μm 2 at least approximately 100 molecules / μm 2 at least approximately 150 molecules / μm 2 at least approximately 200 molecules / μm 2 at least approximately 250 molecules / μm 2 at least approximately 300 molecules / μm 2 at least approximately 350 molecules / μm 2 at least approximately 400 molecules / μm 2 at least approximately 450 molecules / μm 2 at least approximately 500 molecules / μm 2 , at least about 600 molecules / μm 2 at least approximately 700 molecules / μm 2 at least approximately 800 molecules / μm 2 at least approximately 900 molecules / μm 2 , or at least about 1000 molecules / μm 2 It can exist at a density of at least approximately 1000 molecules / μm. CD47 polypeptide (or its fragments) 2 Below, at least approximately 500 molecules / μm 2 The following, or at least about 250 molecules / μm 2CD47 polypeptide (or its fragments) can exist at the following densities: approximately 20 molecules / μm 2 ~about 1000 molecules / μm 2 , about 20 molecules / μm 2 ~about 500 molecules / μm 2 , or approximately 20 molecules / μm 2 ~about 250 molecules / μm 2 It can exist at that density.

[0168] In some embodiments, the amount of CD47 polypeptide (or fragment thereof) may be determined by immunostaining and electron microscopy of CD47, as described in Milani et al. (2019) Science Translational Medicine 11:eaav7325. CD47 polypeptide (or fragment thereof) may be detected in amounts of at least about 10 gold particles per lentivirus particle, at least about 15 gold particles per lentivirus particle, or at least about 20 gold particles per lentivirus particle. CD47 polypeptide (or fragment thereof) may be detected in amounts of at least about 100 or fewer gold particles per lentivirus particle, at least about 80 or fewer gold particles per lentivirus particle, or at least about 60 or fewer gold particles per lentivirus particle. CD47 polypeptide (or fragment thereof) may be detected in amounts of at least about 10 to about 100 gold particles per lentivirus particle, at least about 15 to about 80 gold particles per lentivirus particle, or at least about 20 to about 60 gold particles per lentivirus particle.

[0169] The lentiviral particles of the present invention are CD47 high It may be obtained from producer cells. When used herein, "CD47 high "Producer cells" may refer to producer cells that have increased levels of CD47 (or fragments thereof) on their surface.

[0170] CD47 highProducer cells can be genetically engineered to increase the expression of CD47 (or a fragment thereof) on their cell surface. For example, producer cells may contain a vector encoding CD47 (or a fragment thereof), or they may be edited to introduce a nucleotide sequence encoding CD47 (or a fragment thereof) into their genome. Preferably, producer cells are transduced with a viral vector encoding the CD47 polypeptide (or a fragment thereof).

[0171] CD47 high Producer cells may have a higher concentration of CD47 (or its fragments) on their surface than unmodified producer cells. Preferably, producer cells have 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, or at least about 30 times more CD47 on their cell surface than unmodified producer cells. Preferably, producer cells have about 5 to about 30 times more CD47 (or its fragments) on their cell surface than unmodified producer cells.

[0172] Preferably, the lentiviral particles of the present invention have a higher concentration of CD47 (or its fragments) on their surface than lentiviral particles obtained from unmodified producer cells. Preferably, the lentiviral particles have 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 its fragments) on their surface than lentiviral particles obtained from unmodified producer cells. Preferably, the lentiviral particles have about 5 to about 30 times more CD47 (or its fragments) on their surface than lentiviral particles obtained from unmodified producer cells. CD47 is a member of the immunoglobulin (Ig) superfamily of membrane proteins, and has a single IgV-like domain at its N-terminus, a highly hydrophobic stretch with five transmembrane segments, and an alternatively spliced ​​cytoplasmic C-terminus ranging in length from 3 to 36 amino acids. Mouse, rat, bovine, and human CD47 molecules have been cloned and exhibit approximately 70% overall amino acid identity (see, for example, Brown et al. (2001) Trends Cell Biology 11:130-135).

[0173] The CD47 polypeptide (or fragment thereof) may be a human CD47 polypeptide (or fragment thereof). The CD47 polypeptide may have the amino acid sequence of UniProtKB Q08722.

[0174] Exemplary CD47 polypeptides are provided by SEQ ID NOs. 6-9. Preferably, the CD47 polypeptide contains 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% sequence identity with any of SEQ ID NOs. Preferably, the CD47 polypeptide contains or consists of the amino acid sequence of any of SEQ ID NOs. 6-9.

[0175] An example of the CD47 amino acid sequence is as follows: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFA ILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRNN (Sequence ID 6) MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFG IKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE (Sequence ID 7) MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIV IFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFV (Sequence 8) MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILL FWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLN (Sequence ID 9)

[0176] Exemplary CD47 polypeptides, excluding signal peptides, are provided by SEQ ID NOs: 10-13. Preferably, the CD47 polypeptide contains 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% sequence identity with any of SEQ ID NOs: 10-13. Preferably, the CD47 polypeptide contains or consists of any of the amino acid sequences of SEQ ID NOs: 10-13.

[0177] Examples of CD47 amino acid sequences excluding the signal peptide are as follows: QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFG IKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRNN (Sequence ID 10) QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSG GMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE (Sequence ID 11) QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILL FWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFV (Sequence ID 12) QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKT LKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILAQLLGLVYMKFVASNQKTIQPPRKAVEEPLN (Sequence ID 13)

[0178] Those skilled in the art may, for example, generate variants and / or fragments based on the known structural and functional characteristics of conservative substitutions and / or CD47. These are described, for example, by Fenalti et al. (2021) Nature Communications 12:1-14.

[0179] Preferably, the CD47 and / or CD47 variant fragments retain the ability to inhibit phagocytosis. Preferably, the CD47 fragment and / or CD47 variant may contain the extracellular domain of CD47. The extracellular domain of human CD47 can interact with SIRP-α and inhibit phagocytosis. Optionally, the CD47 fragment and / or CD47 variant may contain the transmembrane domain of CD47. The domains may be linked by interdomain linkers. The fragment and / or variant may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of the full-length CD47 polypeptide.

[0180] Preferably, the variants of sequence number 6 are 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, S 82N, S83T, K85N, K85E, V88A, V88L, V88I, Q90R, L91F, K93N, M100I, M100V, D101G, K10 2R, K102T, S107L, I126F, I127V, K130Q, R132H, S138F, V146I, I150V, I153V, S169A, G1 70R, G170S, G171S, D173Y, I177V, A178G, V181I, V185A, I186V, V188A, I189T, I191V, V198I, A207S, T215I, I219M, Y226C, A231S, T235A, S236F, A240V, A240T, I241V, V243I These may include one or more variations selected from I244T, V246L, Y249F, A252S, A252T, V254A, S257T, I264M, I264L, M266I, M266T, M266V, V287I, V292A, N295S, N295D, Q296L, P302S, N304S, and N304D. These are considered to be acceptable, benign, and / or likely benign variations, as predicted by SIFT, PolyPhen, CADD, REVEL, and MetaLR.

[0181] Preferably, the variants of Sequence ID No. 7 are P3L, A6P, F22L, S27F, F30L, F32Y, T36S, V38L, V38I, F42V, N50S, T51A, T52S, T52A, V56I, R63K, A71T, S75Y, T76A, P78L, P78S, P78A, S82 R, S82N, S83T, K85N, K85E, V88A, V88L, V88I, Q90R, L91F, K93N, M100I, M100V, D101G, K102R, K102T, S107L, I126F, I127V, K130Q, R132H, S138F, V146I, I150 This may include one or more variations selected from V, I153V, S169A, G170R, G170S, G171S, I177V, A178G, V181I, I186V, V188A, I189T, I191V, V198I, A207S, T215I, I219M, Y226C, A231S, T235A, A240V, A240T, I241V, V243I, I244T, V246L, Y249F, A252S, A252T, V254A, I264M, I264L, M266I, M266T, M266V, V287I, V292A, N295S, N295D, and Q296L. These are considered acceptable, benign, and / or likely benign variations, as predicted by SIFT, PolyPhen, CADD, REVEL, and MetaLR.

[0182] An exemplary CD47 fragment is provided by SEQ ID NO: 14. Preferably, the CD47 fragment contains 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% sequence identity with SEQ ID NO: 14. Preferably, the CD47 fragment contains or consists of the amino acid sequence of SEQ ID NO: 14.

[0183] An example of a CD47 fragment is as follows: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSW FSPN (Sequence ID 14)

[0184] One exemplary CD47 fragment, excluding the signal peptide, is provided by SEQ ID NO: 15. Preferably, the CD47 fragment contains 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 with SEQ ID NO: 15. Preferably, the CD47 fragment contains or consists of the amino acid sequence of SEQ ID NO: 15.

[0185] An exemplary CD47 fragment, excluding the signal peptide, is as follows:

[0186] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPN (Sequence ID 15)

[0187] MHC-I low or MHC-I free lentivirus particles The lentiviral particles of the present invention are MHC-I low Lentivirus particles or MHC-I free Lentivirus particles may also be used. In a preferred embodiment, the lentivirus particles of the present invention are MHC-I free These are lentiviral particles.

[0188] As used herein, "MHC-I lowA "lentiviral particle" may refer to a lentiviral particle in which the level of one or more MHC-I molecules on its surface is reduced (i.e., the level of MHC-I molecules exposed on the surface is reduced). The number of MHC-I molecules exposed on the surface may be reduced to a degree that the immune response to MHC-I is therapeutically relevant.

[0189] As used herein, "MHC-I free A "lentiviral particle" may refer to a lentiviral particle that substantially lacks (or does not contain) one or more MHC-I molecules on its surface (i.e., substantially lacks (or does not contain) MHC-I molecules exposed on its surface). Specifically, the surface of a lentiviral particle does not need to contain MHC-I.

[0190] Major histocompatibility complex class I (MHC-I) is a heterodimeric membrane protein presented on the outer lobe of the cell membrane (see, e.g., Penn et al. (2005) Major histocompatibility complex (MHC).eLS). MHC-I functions to bind and present peptide fragments of proteins to the extracellular environment, where these peptide fragments can be recognized by CD8+ cytotoxic T cells. Peptide fragments produced from normal cellular proteins do not activate cytotoxic T cells due to central and peripheral tolerance mechanisms. However, exogenous peptides (e.g., those derived from viral proteins) can trigger an immune response and destroy cells. Allogeneic MHC-I proteins themselves can be recognized by the immune system. For example, antibodies can directly bind to MHC-I epitopes. As a result, lentiviral particles containing MHC-I molecules from allogeneic sources can be targeted and neutralized by the immune system.

[0191] The term "MHC-I molecule" may refer to the human MHC-I molecule. Human MHC-I, also known as human leukocyte antigen class I (HLA-I), is expressed on almost all nucleated cells. HLA-I consists 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 linked non-covalently. The HLA-I α chain is polymorphic. Six HLA-I α chains have been identified to date, including three classical, highly polymorphic α chains (HLA-A, HLA-B, and HLA-C) and three non-classical, less polymorphic α chains (HLA-E, HLA-F, and HLA-G). The MHC-I molecule may contain, or consist of, HLA-A, HLA-B, and HLA-C molecules, each containing an invariant β2M sequence.

[0192] The term "MHC-I molecule" may also include variant MHC-I sequences, such as polymorphisms of the HLA-Iα chain sequence and / or β2M sequence. For example, variant MHC-I sequences may include HLA-Iα chain sequences and / or β2M sequences having single nucleotide polymorphisms (SNPs) or multiple SNPs.

[0193] Any suitable analytical method may be used to quantify the amount of MHC-I molecules present on the surface of lentiviral particles.

[0194] In some embodiments, the amount of MHC-I molecules may be determined by immunostaining and electron microscopy for MHC-I, as described, for example, in Milani et al. (2017) EMBO Molecular Medicine 9:1558-1573. MHC-I molecules may be detected in amounts of less than 10 gold particles per lentivirus particle, less than 9 gold particles per lentivirus particle, less than 8 gold particles per lentivirus particle, less than 7 gold particles per lentivirus particle, less than 6 gold particles per lentivirus particle, less than 5 gold particles per lentivirus particle, less than 4 gold particles per lentivirus particle, less than 3 gold particles per lentivirus particle, less than 2 gold particles per lentivirus particle, less than 1 gold particle per lentivirus particle, or about 0 gold particles per lentivirus particle. MHC-I molecules may be undetectable (e.g., the amount of gold particles detected may not be significantly higher than the background level).

[0195] The lentiviral particles of the present invention are MHC-I low Producer cells or MHC-I free They may be obtained from producer cells. In a preferred embodiment, the lentiviral particles of the present invention are MHC-I free Obtained from producer cells. When used herein, "MHC-I low "Producer cells" may refer to producer cells that have reduced levels of one or more MHC-I molecules on their surface.

[0196] As used herein, "MHC-I free A "producer cell" may refer to a producer cell that substantially lacks or does not contain one or more MHC-I molecules on its surface. Specifically, the surface of a lentiviral particle may not contain MHC-I, for example.

[0197] MHC-I low or MHC-I freeProducer cells can be genetically engineered to reduce MHC-I expression on their cell surface. For example, the cells may have genetically engineered disruptions of the gene encoding β2-microglobulin and / or the gene encoding the MHC-Iα chain.

[0198] Methods of genetic engineering to reduce protein expression are known in the art. For example, this can be achieved by target gene knockout. To reduce protein expression, the gene encoding the protein itself or its regulatory sequence (e.g., its promoter) may be knocked out. Knockout can be achieved by deletion of a portion of the coding nucleic acid sequence, which may involve deletion of a portion of the protein essential for expression or stability, alteration of the reading frame of the coding sequence, or by base editing. Preferred methods for target gene knockout include the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas-based RNA guide nucleases (see, for example, Gaj et al. (2013) Trends Biotechnol 31:397-405). For example, a CRISPR / Cas9 RNA-guided nuclease can be used to catalyze double-strand breaks at a specific gene locus in the genome, provided that a suitable RNA guide designed to bind to that locus is provided. Cas9 and the guide RNA can be delivered to the target cell by translocation of a protein- and RNA-encoding vector. 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 often inserts random nucleotides and disrupts the reading frame of the target gene. Alternatively, genetic manipulation to reduce protein expression can be achieved using RNAi techniques, microRNA, or antisense RNA to suppress the expression of the target gene.

[0199] After performing targeted gene knockout or expression suppression, the resulting cell population can be screened to select and enrich cells exhibiting the desired phenotype, such as reduced expression of surface-exposed MHC-I. Suitable techniques for screening and enrichment, which are known in the art, include flow cytometry and fluorescence-activated cell sorting (FACS).

[0200] In some embodiments, producer cells include genetically engineered disruption of the gene encoding β2-microglobulin. β2-microglobulin stabilizes MHC-I, and therefore, cells lacking β2-microglobulin exhibit reduced MHC-I expression on the cell surface. The cells may include genetically engineered disruption in all copies of the gene encoding β2-microglobulin.

[0201] In another embodiment, the cell includes a genetically engineered disruption of one or more genes encoding the MHC-Iα chain. The cell may include a genetically engineered disruption of all copies of the gene encoding the MHC-Iα chain.

[0202] The cells may include both genetically modified disruptions of the gene encoding β2-microglobulin and genetically modified disruptions of the gene encoding the MHC-Iα chain.

[0203] A reduction in MHC-I expression on the cell surface may refer to a reduction in the number of MHC-I molecules expressed on the surface of genetically modified cells compared to the number of MHC-I molecules expressed on the surface of cells without genetic manipulation but otherwise substantially identical conditions. MHC-I expression on the cell surface may be reduced so that the number of surface-exposed MHC-I molecules is, for example, less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the number of surface-exposed MHC-I molecules presented in the absence of genetic manipulation. In some embodiments, MHC-I expression on the cell surface is reduced so that the number of surface-exposed MHC-I molecules is 0% of the number of surface-exposed MHC-I molecules presented in the absence of genetic manipulation.

[0204] The expression of MHC-I on the cell surface is preferably reduced to such an extent that the cells substantially lack MHC-I molecules exposed on their surface. In this context, "substantially lacking" may mean that the number of MHC-I molecules expressed on the surface of genetically modified cells is substantially reduced compared to the number of MHC-I molecules expressed on the surface of non-genetically modified cells, thereby reducing the immune response to MHC-I on lentiviral particles produced by the cells to a therapeutically useful degree.

[0205] Preferably, the lentiviral particles of the present invention have a lower concentration of MHC-I molecules on their surface than lentiviral particles obtained from unmodified producer cells. Preferably, the lentiviral particles have about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or less than 1% of the number of surface-exposed MHC-I molecules presented on lentiviral particles obtained from unmodified producer cells. In some embodiments, the lentiviral particles have about 20% or less of the number of surface-exposed MHC-I molecules presented on lentiviral particles obtained from unmodified producer cells.

[0206] In some embodiments, the lentiviral particles of the present invention substantially lack MHC-I molecules on their surface. In this context, "substantially lacking" may mean that there is no detectable immune response due to molecules on the surface of the lentiviral particles.

[0207] In some embodiments, the lentiviral particles of the present invention do not contain MHC-I molecules on their surface. In this context, “does not contain” may mean that no detectable molecules are present on the surface of the lentiviral particles (e.g., by immunostaining and electron microscopy). As used herein, “undetectable” may mean a level that is not statistically significant compared to background levels.

[0208] In some embodiments, the lentiviral particles of the present invention have reduced HLA-A, HLA-B, and / or HLA-C molecules on their surface. Preferably, the lentiviral particles have about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or less than 1% of the number of surface-exposed HLA-A molecules presented on lentiviral particles obtained from unmodified producer cells. Preferably, the lentiviral particles have about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or less than 1% of the number of surface-exposed HLA-B molecules presented on lentiviral particles obtained from unmodified producer cells. Preferably, the lentiviral particles have about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or less than 1% of the number of surface-exposed HLA-C molecules presented on the lentiviral particles obtained from unmodified producer cells.

[0209] In some embodiments, the lentiviral particles of the present invention substantially lack HLA-A, HLA-B, and / or HLA-C molecules on their surface. In some embodiments, the lentiviral particles of the present invention substantially lack HLA-A, HLA-B, and HLA-C molecules on their surface. In some embodiments, the lentiviral particles of the present invention do not contain HLA-A, HLA-B, and / or HLA-C molecules on their surface. In some embodiments, the lentiviral particles of the present invention do not contain HLA-A, HLA-B, and HLA-C molecules on their surface. As described above, the HLA-I molecule consists of two polypeptide chains, an HLA-I heavy chain (α chain) and a β2 microglobulin (β2M or β chain). The HLA-I α chain and β2M are linked non-covalently.

[0210] Those skilled in the art will be able to easily determine the amino acid and nucleic acid sequences of the HLA-Iα chain. For example, the HLA-Iα chain can be identified in the genome sequence using its position within the region of the major histocompatibility complex of the chromosome (see, e.g., Penn et al. (2005) Major histocompatibility complex (MHC).eLS).

[0211] The HLA-Aα chain may have the amino acid sequence of UniProtKB P04439. Exemplary HLA-Aα chains are provided by SEQ ID NOs. 16 and 17. Preferably, the HLA-Aα chain contains 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% sequence identity with SEQ ID NOs. 16 or 17. Preferably, the HLA-Aα chain contains or consists of the amino acid sequence of SEQ ID NOs. 16 or 17. MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRK WEAAHEAEQLRAYLDGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRK GGSYTQAASSDSAQGSDVSLTACKV (Sequence ID 16) MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRK WEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGE GVKDRKGGSYTQAASSDSAQGSDVSLTACKV (Sequence ID 17)

[0212] The HLA-Bα chain may have the amino acid sequence of UniProtKB P01889. An exemplary HLA-Bα chain is provided by SEQ ID NO: 18. Preferably, the HLA-Bα chain contains 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% sequence identity with SEQ ID NO: 18. Preferably, the HLA-Bα chain contains or consists of the amino acid sequence of SEQ ID NO: 18. MLVMAPRTVLLLLSAALALTETWAGSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRR AYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSYSQAACSDSAQGSDVSLTA (Sequence No. 18)

[0213] The HLA-Cα chain may have the amino acid sequence of UniProtKB P10321. Exemplary HLA-Cα chains are provided by SEQ ID NOs. 19 and 20. Preferably, the HLA-Cα chain contains 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% sequence identity with SEQ ID NOs. 19 or 20. Preferably, the HLA-Cα chain contains or consists of the amino acid sequence of SEQ ID NOs. 19 or 20. MRVMAPRALLLLLSGGLALTETWACSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAY LEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEPSSQPTIPIMGIVAGLAVLVVLAVLGAVVTAMMCRRKSSGGKGGSCSQAACSNSAQGSDESLITCKA (Sequence ID 19) MRVMAPRALLLLLSGGLALTETWACSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNEHLRSCTAADTA AQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLRWGGKGGSCSQAACSNSAQGSDESLITCKA (Sequence ID 20)

[0214] The amino acid and nucleic acid sequences encoding β2M are also publicly known in the art. For example, the nucleic acid sequence of human β2M is deposited in GenBank under accession number NM_004048.

[0215] The HLAβ chain may be that of UniProtKB P61769. An exemplary HLAβ chain is provided by SEQ ID NO: 21. Preferably, the HLAβ chain contains 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% sequence identity with respect to SEQ ID NO: 21. Preferably, the HLAβ chain contains or consists of the amino acid sequence of SEQ ID NO: 21. MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVVTNHLSQPKIVKWDRDM (Sequence ID 21)

[0216] The lentiviral particles of the present invention are CD47 high / MHC-I free Lentivirus particles or CD47 high / MHC-I low Lentivirus particles may also be used. In a preferred embodiment, the lentivirus particles of the present invention are CD47 high / MHC-I free These are lentiviral particles.

[0217] The lentiviral particles of the present invention are CD47 high / MHC-I free Producer cells or CD47 high / MHC-I low It can be obtained from producer cells. In a preferred embodiment, the lentiviral particles of the present invention are CD47 high / MHC-I free It is obtained from producer cells.

[0218] Enveloped virus-like particles (VLPs) Virus-like particles (VLPs) are very similar to viruses but do not contain viral genetic material. VLPs can be synthesized via the expression of viral structural proteins, which can then self-assemble into virus-like particles. VLPs may retain the ability to transduce cells and release cargo. The disclosures herein regarding enveloped virus particles may also apply to enveloped VLPs where appropriate in the context.

[0219] VLPs can originate from enveloped viruses such as lentiviruses.

[0220] VLPs can be used to deliver protein cargo such as Cas9 nuclease or ribonucleoprotein (RNP). VLPs can also be used to deliver one or more components of gene editing mechanisms (e.g., CRISPR / Cas9, base editors, or prime editors).

[0221] Adjustment element The enveloped virus particles (e.g., lentiviral particles) of the present invention may further comprise one or more regulatory elements that can act pre-transcriptionally or post-transcriptionally. Preferably, the protein coding sequence is operably linked to one or more regulatory elements that can act pre-transcriptionally or post-transcriptionally. One or more regulatory elements can promote protein expression in hepatocytes (e.g., hepatocytes).

[0222] As used herein, “regulatory element” may refer to any nucleotide sequence that promotes polypeptide expression, for example, by increasing transcript expression or enhancing mRNA stability. Preferred regulatory elements include, for example, promoters, enhancer elements, post-transcriptional regulatory elements, polyadenylation sites, and Kozak sequences.

[0223] promoter The enveloped virus particles (e.g., lentiviral particles) of the present invention may include a promoter such as a liver-specific (e.g., hepatocyte-specific) promoter. Preferably, the protein coding sequence is operably linked to the promoter such as a liver-specific (e.g., hepatocyte-specific) promoter.

[0224] A "promoter" can refer to a region of DNA that initiates the transcription of a gene. Promoters are located near the transcription start site of a gene.

[0225] As used herein, “tissue-specific promoter” may refer to a promoter that preferentially promotes the expression of a transgene in a particular type of cell or tissue. Preferably, a tissue-specific promoter can promote higher expression of a transgene in one cell type compared to other cell types. Higher expression can be measured, for example, by measuring the expression of a transgene operably linked to a promoter, such as green fluorescence protein (GFP), and the expression of the transgene correlates with the promoter’s ability to promote gene expression. For example, a tissue-specific promoter may be a promoter that promotes a transgene expression level in one cell type that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% higher than the expression level in other cell types.

[0226] In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the promoter is a hepatocyte-specific promoter.

[0227] Preferably, the promoter may be a promoter associated with (or derived from) a gene that exhibits selective expression in human hepatocytes (e.g., hepatocytes). Methods for identifying gene-associated promoters are well known to those skilled in the art.

[0228] Exemplary liver-specific and / or hepatocyte-specific promoters are described in Kattenhorn et al. (2016) Human Gene Therapy 27:947-961 and include the transthyretin (TTR) promoter, the alpha-1-antyrpsin (AAT) promoter, the thyroxine-binding globulin (TBG) promoter, the APoE / hAAT promoter, the HCR-hAAT promoter, the LP1 promoter, and the HLP promoter.

[0229] Manipulated promoter variants derived from any of these promoters can be used, provided that the variants retain the ability to drive liver-specific and / or hepatocyte-specific expression of the transgene functionally bound to the promoter. Those skilled in the art will be able to obtain such variants using methods known in the art. The variants may have, for example, 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% sequence identity with respect to any of the promoters.

[0230] Any fragment of these promoters (or their variants) may be used, provided that the fragment retains the ability to drive liver-specific and / or hepatocyte-specific expression of the transgene functionally bound to the promoter. Those skilled in the art will be able to obtain such fragments using methods known in the art. The fragments 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 in length.

[0231] In some embodiments, the promoter is selected from the group consisting of the transthyretin (TTR) promoter, the α1-antitrypsin (AAT) promoter, the thyroxine-binding globulin (TBG) promoter, the APoE / hAAT promoter, the HCR-hAAT promoter, the LP1 promoter, and the HLP promoter.

[0232] In some embodiments, the promoter is a TTR promoter, or a variant and / or fragment thereof. In some embodiments, the promoter is an enhanced TTR (ET) promoter, or a variant and / or fragment thereof.

[0233] The promoter may be a constitutive promoter. As used herein, “constitutive promoter” is always an active promoter.

[0234] Alternatively, the promoter may be an inductive promoter. As used herein, “inductive promoter” is a promoter that is active only under specific conditions. For example, the expression of a transgene may be induced by a small molecule or drug (e.g., bound to a promoter, regulatory sequence, or transcriptional repressor or activator molecule), or by using an environmental trigger. Types of inductive promoters include chemically inductive promoters (e.g., Tet-on systems), temperature-inductive promoters (e.g., Hsp70-derived or Hsp90-derived promoters), and photo-inductive promoters. Preferably, the promoter is a chemically inductive promoter. Any preferred method for manipulating the inductive promoter may be used.

[0235] Enhancer Element The enveloped virus particles (e.g., lentiviral particles) of the present invention may contain enhancers such as liver-specific (e.g., hepatocyte-specific) enhancers. Preferably, the protein coding sequence is operably linked to the enhancer such as the liver-specific (e.g., hepatocyte-specific) enhancer.

[0236] An "enhancer" or "enhancer element" may refer to a region of DNA that can be bound by a protein (activator) to increase the likelihood of transcription of a particular gene occurring. Enhancers are cis-acting. Enhancers can be spaced up to 1 Mbp (1,000,000 bp) away from the gene and may be located upstream or downstream of the start site.

[0237] As used herein, an "organ-specific enhancer" is an enhancer that preferentially promotes the expression of a gene in a particular cell or tissue. Preferably, an organ-specific enhancer can promote higher expression of a gene in a particular cell type as compared to other cell types. Higher expression can be measured, for example, by measuring the expression of a transgene operably linked to the enhancer, such as green fluorescent protein (GFP), and the expression of the transgene correlates with the ability of the enhancer to promote the expression of the gene. For example, an organ-specific enhancer can be an enhancer that promotes the expression level of a gene that is 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 in a particular cell type as compared to the expression level in other cell types.

[0238] Suitable organ-specific enhancers are well known to those skilled in the art. The enhancer may be a liver-specific enhancer, preferably a hepatocyte-specific enhancer.

[0239] Preferably, the enhancer can be (or can be derived from) an enhancer associated with a gene having selective expression in human hepatocytes (e.g., hepatocytes). Preferably, the enhancer can be (or can be derived from) an enhancer associated with a gene having selective expression in human hepatocytes. Methods for identifying enhancer regions associated with a gene are well known to those skilled in the art.

[0240] Exemplary liver-specific enhancers and / or hepatocyte-specific enhancers are described in Kramer et al. (2003) Molecular Therapy 7:375-385, and include enhancer regions of albumin, α1-antitrypsin, hepatitis B virus core protein, and hemopexin genes. Other liver-specific enhancers and / or hepatocyte-specific enhancers include apolipoprotein E (APoE) enhancer, hepatic control region (HCR) enhancer, and α1-antitrypsin (AAT) enhancer.

[0241] Engineered enhancer variants derived from any of these enhancers may be used, provided that the variant retains the ability to drive liver-specific and / or hepatocyte-specific expression of a transgene operably linked to the enhancer. Those skilled in the art 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% sequence identity to any of the enhancers.

[0242] 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 linked to the enhancer. Those skilled in the art will be able to arrive at such fragments using methods known in the art. 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 in length.

[0243] The enveloped virus particles (e.g., lentiviral particles) of the present invention may include a liver-specific promoter and / or a liver-specific enhancer. Preferably, the protein-coding sequence is operably linked to the liver-specific promoter and / or enhancer. Preferably, the protein-coding sequence is operably linked to a hepatocyte-specific promoter and / or enhancer. The promoter and enhancer may be any combination of the above, for example, an hAAT promoter and an ApoE enhancer or an HCR enhancer.

[0244] Post-transfer regulatory elements The enveloped virus particles (e.g., lentiviral particles) of the present invention may include one or more additional post-transcriptional regulatory elements (in addition to, for example, one or more miRNA target sequences). Preferably, the protein-coding sequences include one or more It is operably linked to further post-transcriptional regulatory elements. These further post-transcriptional regulatory elements may improve gene expression.

[0245] The enveloped virus particles (e.g., lentiviral particles) of the present invention may contain a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). Preferably, a protein-coding sequence is operably linked to the WPRE.

[0246] Suitable WPRE sequences are well known to those skilled in the art (see, for example, Zufferey et al. (1999) Journal of Virology 73:2886-2892; Zanta-Boussif et al. (2009) Gene Therapy 16:605-619). Preferably, the WPRE is either a wild-type WPRE or a mutant WPRE. For example, a WPRE may be mutated to suppress the translation of woodchuck hepatitis virus X protein (WHX) by, for example, mutating the WHX ORF translation start codon.

[0247] In some embodiments, the WPRE comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with respect to SEQ ID NO: 22. Preferably, the WPRE comprises or consists of a nucleotide sequence or fragment thereof having 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% sequence identity with respect to SEQ ID NO: 22.

[0248] In some embodiments, WPRE includes or consists of the nucleotide sequence of SEQ ID NO: 22 or a fragment thereof. AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCA CTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC CCTCAATCCAGCGGACCTTCCTTCCCGC (Sequence ID 22)

[0249] Polyadenylated sequence The enveloped virus particles (e.g., lentiviral particles) of the present invention may contain a polyadenylated sequence. Preferably, a protein-coding sequence is operably linked to the polyadenylated sequence. The polyadenylated sequence may be inserted after the protein-coding sequence to improve transgene expression.

[0250] A polyadenylated sequence typically includes a polyadenylation signal, a polyadenylation site, and a downstream element. The polyadenylation signal contains a sequence motif recognized by the RNA cleavage complex; the polyadenylation site is the cleavage site where a poly-A tail is attached to the mRNA; and the downstream element is a GT-rich region, usually located immediately downstream of the polyadenylation site, which is important for efficient processing.

[0251] Suitable polyadenylation sequences are well known to those skilled in the art (see, for example, Schambach et al. (2007) Molecular Therapy 15:1167-1173; Choi et al. (2014) Molecular Brain 7:1-10). Exemplary polyadenylation sequences include the bGH poly(A) signaling sequence and the SV40pA signaling sequence.

[0252] Kozak Array The enveloped virus particles (e.g., lentiviral particles) of the present invention may contain a Kossack sequence. Preferably, a protein-coding sequence is operably linked to the Kossack sequence. To improve translation initiation, the Kossack sequence may be inserted before the start codon.

[0253] Suitable Kozak sequences are well known to those skilled in the art (see, for example, Kozak (1987) Nucleic Acids Research 15:8125-8148).

[0254] In some embodiments, the Kozak sequence comprises or consists of a nucleotide sequence or fragment thereof having at least 80% sequence identity with SEQ ID NO: 23.

[0255] In some embodiments, the Kozak sequence includes or consists of the nucleotide sequence of sequence number 23 or a fragment thereof. GCCACC (Sequence No. 23)

[0256] Other cis-acting elements The enveloped virus particles of the present invention (e.g., lentiviral particles) may include any other suitable cis-acting elements, such as a rev response element (RRE), a retroviral psi packaging element, a primer binding site (PBS), a TAT activation region (TAR), a splice donor site and a splice acceptor site, and one or more of a central polyprint lactulose and a terminal polyprint lactulose.

[0257] Long terminal repeat (LTR) The envelope virus particles of the present invention (e.g., lentivirus particles) may contain one or more long terminal repeats (LTRs). The LTR is responsible for provirus integration and transcription. Typically, naturally occurring LTRs include a U3 region, an R region, and a U5 region.

[0258] Envelope virus particles (e.g., lentivirus particles) may contain a 5' LTR and / or a 3' LTR. Envelope virus particles (e.g., lentivirus particles) may contain a 5' LTR and a 3' LTR. Preferably, the 5' LTR includes an R region and a U5 region, and optionally includes a U3 region. Preferably, the 3' LTR includes a U3 region, an R region, and a U5 region.

[0259] Suitable LTR sequences are well known to those skilled in the art (see, for example, Frech et al. (1996) Virology 224:256-267).

[0260] In some embodiments, the LTR comprises or consists of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 24 or a fragment thereof. Preferably, the LTR comprises or consists of a nucleotide sequence having 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% sequence identity to SEQ ID NO: 24 or a fragment thereof.

[0261] In some embodiments, the LTR comprises or consists of the nucleotide sequence of SEQ ID NO: 24 or a fragment thereof. TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG (Sequence ID 24)

[0262] The enveloped virus particles (e.g., lentiviral particles) of the present invention may contain one or more self-inactivating long terminal repeats (SIN-LTRs). A "SIN-LTR" may include a deletion that disables full-length virus transcription after integration into a host cell. For example, a 3'SIN-LTR may include a deletion in the U3 region that removes a promoter / enhancer element (see, e.g., Zufferey et al. (1998) Journal of Virology 72:9873-9880). This deletion is copied to a 5'LTR after reverse transcription, thereby resulting in gene expression in a target cell that depends on a selected internal promoter.

[0263] Suitable SIN-LTR sequences are well known to those skilled in the art (see, for example, Zufferey et al. (1998) Journal of Virology 72:9873-9880; Miyoshi et al. (1998) Journal of Virology 72:8150-8157).

[0264] In some embodiments, the 5'LTR comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 25. Preferably, the 5'LTR comprises or consists of a nucleotide sequence or fragment thereof having 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% sequence identity with SEQ ID NO: 25.

[0265] In some embodiments, the 5'LTR includes or consists of the nucleotide sequence of SEQ ID NO: 25 or a fragment thereof. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG (Sequence ID 25)

[0266] In some embodiments, the 5'LTR and / or 3'LTR contain or consist of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 24. Preferably, the 5'LTR and / or 3'LTR contain or consist of a nucleotide sequence or fragment thereof having 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% sequence identity with SEQ ID NO: 24. In some embodiments, the 5'LTR and / or 3'LTR contain or consist of the nucleotide sequence or fragment thereof of SEQ ID NO: 24.

[0267] In some embodiments, the 5'LTR and / or 3'LTR comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 24. Preferably, the 5'LTR and / or 3'LTR comprises or consists of a nucleotide sequence or fragment thereof having 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% sequence identity with SEQ ID NO: 24.

[0268] In some embodiments, the 5'LTR and / or 3'LTR include or consist of the nucleotide sequence of SEQ ID NO: 24 or a fragment thereof.

[0269] Primer binding site (PBS) The enveloped virus particles (e.g., lentiviral particles) of the present invention may include a primer-binding site (PBS). The PBS is a cis-acting element to which a primer can bind and initiate reverse transcription of the RNA genome (see, for example, Lanchy et al. (1998) Journal of Biological Chemistry 273:24425-24432).

[0270] Suitable retrovirus PBSs are well known to those skilled in the art.

[0271] In some embodiments, PBS comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 26. Preferably, PBS comprises or consists of a nucleotide sequence or 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: 26.

[0272] In some embodiments, PBS comprises or consists of the nucleotide sequence or fragment thereof of SEQ ID NO: 26. TGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAG (Sequence ID 26)

[0273] Retrovirus psi packaging element The enveloped virus particles (e.g., lentiviral particles) of the present invention may include retroviral psi packaging elements. Retroviral psi packaging elements are cis-acting elements involved in regulating the process of packaging the retroviral RNA genome into the viral capsid during replication (see, for example, McBride et al. (1997) Journal of Virology 71:4544-4554). The retroviral psi packaging elements may form part of the 5' region of the gag gene. Suitable retroviral psi packaging elements are well known to those skilled in the art.

[0274] In some embodiments, the retroviral psi packaging element comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 27. Preferably, the retroviral psi packaging element comprises or consists of a nucleotide sequence or fragment thereof having 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% sequence identity with SEQ ID NO: 27.

[0275] In some embodiments, the retroviral psi packaging element includes or consists of the nucleotide sequence of SEQ ID NO: 27 or a fragment thereof. ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATA CTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGAT (Sequence ID 27)

[0276] Rev response element (RRE) The enveloped virus particles of the present invention (e.g., lentiviral particles) may include a rev response element (RRE). The RRE is a cis-acting element that enables the efficient transport of the incorporated proviral RNA transcript from the nucleus to the cytoplasm of an infected target cell (see, for example, Pollard et al. (1998) Annual Review of Microbiology 52:491-532).

[0277] Suitable RRE sequences are well known to those skilled in the art.

[0278] In some embodiments, the RRE comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 28. Preferably, the RRE comprises or consists of a nucleotide sequence or 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: 28.

[0279] In some embodiments, the RRE includes or consists of the nucleotide sequence of sequence number 28 or a fragment thereof. GGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTT (Sequence No. 28)

[0280] Central polypurine sequence (cPPT) The enveloped virus particles (e.g., lentiviral particles) of the present invention may contain a central polypurine tract (cPPT). The cPPT may enable the initiation of positive-chain synthesis (see, for example, Follenzi et al. (2000) Nature Genetics 25:217-222).

[0281] Suitable cPPT sequences are well known to those skilled in the art.

[0282] In some embodiments, the cPPT comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 29. Preferably, the cPPT comprises or consists of a nucleotide sequence or fragment thereof having 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% sequence identity with SEQ ID NO: 29.

[0283] In some embodiments, cPPT comprises or consists of the nucleotide sequence of SEQ ID NO: 29 or a fragment thereof. AACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATC (Sequence ID 29)

[0284] Target nucleotide The viral particles of the present invention may, for example, contain a transgene encoding a target protein. Preferably, the transgene produces a therapeutic effect.

[0285] Suitable transgenes include, but are not limited to, sequences encoding enzymes, cytokines, chemokines, hormones, antibodies, antioxidant molecules, engineered immunoglobulin-like molecules, single-chain antibodies, fusion proteins, immunocostimulatory molecules, immunomodulatory molecules, antisense RNA, microRNA, shRNA, siRNA, ribozymes, miRNA target sequences, transdomain-negative variants of target proteins, toxins, conditional toxins, antigens, tumor suppressor proteins, growth factors, transcription factors, membrane proteins, surface receptors, anticancer molecules, vasoactive proteins and peptides, antiviral proteins and antiviral ribozymes, and their derivatives (e.g., derivatives having the relevant reporter group). Transgenes may also encode prodrug-activating enzymes. Examples of transgenes include coagulation factor VIII or coagulation factor IX or their engineered derivatives, which may be used in gene therapy for hemophilia, or β-globin chains, which may be used in gene therapy for thalassemia / sickle cell disease.

[0286] The introduced gene may encode, for example, a chimeric antigen receptor (CAR).

[0287] The transgene may encode LDLR. In some embodiments, enveloped virus particles (e.g., lentiviral particles) contain a nucleotide sequence encoding LDLR.

[0288] In some embodiments, the LDLR comprises or consists of an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 5.

[0289] In some embodiments, PCSK9 comprises or consists of the amino acid sequence of SEQ ID NO: 5 or a fragment thereof.

[0290] An example of a nucleotide sequence encoding LDLR is as follows: (Sequence ID 33, Human LDLR)

[0291] In some embodiments, the nucleotide sequence encoding the LDLR comprises or consists of a nucleotide sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to sequence number 33.

[0292] In some embodiments, the nucleotide sequence encoding the LDLR includes or consists of the nucleotide sequence of sequence number 33 or a fragment thereof.

[0293] Pharmaceutical composition The enveloped virus particles or transduced cells of the present invention can be formulated for administration to a subject together with a pharmaceutically acceptable carrier, diluent, or excipient. The enveloped VLPs of the present invention can be formulated for administration to a subject together with a pharmaceutically acceptable carrier, diluent, or excipient. Suitable carriers and diluents include isotonic salines, such as phosphate-buffered saline, which may contain human serum albumin.

[0294] The handling of cell therapy products is preferably carried out in accordance with the FACT-JACIE international standard for cell therapy.

[0295] The pharmaceutical composition may contain or consist of a therapeutically effective amount of a pharmaceutically active agent (e.g., enveloped virus particles). The pharmaceutical composition preferably contains a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).

[0296] "Pharmacologically acceptable" includes the fact that the formulation is sterile and free of pyrogens. The carrier, diluent, and / or excipient must be "acceptable" in the sense that it is compatible with enveloped virus particles and is not harmful to its recipient. Typically, the carrier, diluent, and excipient are sterile, pyrogen-free saline or infusion medium, but other acceptable carriers, diluents, and excipients may be used.

[0297] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the field of pharmacy. The choice of carrier, excipient, or diluent for pharmaceutical use can be made in relation to the intended method of administration and standard pharmaceutical practice. Pharmaceutical compositions may contain, (or in addition to) any suitable binder(s), lubricants(s), suspending agents(s), coating agents(s), or solubilizers(s) as carriers, excipients, or diluents.

[0298] Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, fragrance oils, fatty acid monoglycerides and fatty acid diglycerides, petroleum ether fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0299] The enveloped virus particles, cells, or pharmaceutical compositions according to the present invention may be administered in an appropriate manner to treat and / or prevent the diseases described herein. The enveloped VLPs according to the present invention may be administered in an appropriate manner to treat and / or prevent the diseases described herein. Preferred administration methods are known to those skilled in the art.

[0300] The dosage and frequency of administration can be determined by those skilled in the art, for example, depending on factors such as the condition of the subject and the type and severity of the disease. The pharmaceutical composition can be formulated accordingly. The enveloped virus particles, cells, 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 enveloped virus particles, cells, or pharmaceutical composition can be administered in the form of a sterile aqueous solution that may contain other substances, such as salts or glucose sufficient to make the solution isotonic with blood. The aqueous solution may preferably be buffered (preferably to a pH of 3-9). The pharmaceutical composition can be formulated accordingly. Preparation of suitable parenteral formulations under sterile conditions is readily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0301] The enveloped virus particles, cells, or pharmaceutical compositions according to the present invention can be administered systemically, for example, by intravenous or intraperitoneal injection. In some embodiments, the enveloped virus particles, cells, or pharmaceutical compositions according to the present invention are administered by intravenous injection. The pharmaceutical compositions can be formulated accordingly.

[0302] The enveloped virus particles, cells, or pharmaceutical compositions according to the present invention may be administered topically, for example, by direct injection, intra-arterial injection, or intra-portal injection. In some embodiments, the enveloped virus particles, cells, or pharmaceutical compositions according to the present invention are administered topically to the liver. In some embodiments, the enveloped virus particles, cells, or pharmaceutical compositions according to the present invention are administered by intrahepatic injection, intrahepatic arterial injection, or intra-portal injection. The pharmaceutical compositions may be formulated accordingly.

[0303] The pharmaceutical composition may contain enveloped virus particles or cells of the present invention in an infusion medium, for example, in a sterile isotonic solution. The pharmaceutical composition may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0304] Enveloped virus particles, cells, or pharmaceutical compositions may be administered in single or multiple doses. Preferably, enveloped virus particles, cells, or pharmaceutical compositions may be administered in a single, one-time dose. Pharmaceutical compositions may be formulated accordingly.

[0305] Enveloped virus particles, cells, or pharmaceutical compositions may be administered in various doses (e.g., measured in transducing units (TU) per kilogram). In any case, a physician can determine the most suitable actual dose for each individual subject, and the dose may vary depending, for example, on the age, weight, and response of a particular subject. Pharmaceutical compositions may be formulated accordingly.

[0306] Enveloped virus particles, cells, or pharmaceutical compositions may be administered to any subject that requires them. The subject may be a mammal (e.g., human).

[0307] The enveloped virus particles or enveloped VLPs of the present invention can be used to transduce cells in vitro. The transduced cells can then be administered to a target.

[0308] In another embodiment, the present invention provides a method for transducing cells, comprising contacting the cells with the enveloped virus particles or enveloped VLPs of the present invention. This method may be, for example, an in vitro or in vitro method.

[0309] Variants, derivatives, analogues, and fragments In addition to the specific polypeptides and polynucleotides referred to herein, the present invention also encompasses their variants, derivatives, and fragments.

[0310] In the context of the present invention, a “variant” of any given sequence is a sequence in which a specific sequence of residues (either amino acid residues or nucleic acid residues) is modified in such a way that the polypeptide or polynucleotide retains at least one or all of its endogenous functions. Variant sequences can be obtained by the addition, deletion, substitution, modification, exchange and / or alteration of at least one residue present in a naturally occurring polypeptide or polynucleotide.

[0311] When used herein in relation to the proteins or polypeptides of the present invention, the term “derivative” includes any substitution, variation, modification, exchange, deletion and / or addition of one (or more) amino acid residues to or from a sequence, provided that the resulting protein or polypeptide retains at least one or all of its endogenous functions.

[0312] Typically, amino acid substitutions can consist of, for example, one, two, or three to ten or twenty substitutions, provided that the modified sequence retains the required activity or capability. Amino acid substitutions may involve the use of non-natural analogs.

[0313] The polypeptides used in the present invention may also undergo silent changes, including deletions, insertions, or substitutions of amino acid residues that result in functionally equivalent polypeptides. Planned amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as the endogenous function is preserved. 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 having similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0314] Conservative substitutions can be performed, for example, according to the following table. Amino acids in the same block in the second column and the same row in the third column can be substituted for each other.

[0315] [Table 1]

[0316] The effects of addition, deletion, substitution, modification, exchange, and / or variation can be predicted using any suitable predictive tool, e.g., SIFT (Vaser et al. (2016) Nature Protocols 11:1-9), PolyPhen-2 (Adzhubei et al. (2013) Current Protocols in Human Genetics 76:7-20), CADD (Rentzsch et al. (2021) Genome Medicine 13:1-12), REVEL (Ioannidis et al. (2016) The American Journal of Human Genetics 99:877-885), MetaLR (Dong et al. (2015) Human Molecular Genetics 24:2125-2137) and / or MutationAssessor (Reva et al. (2011) Nucleic Acids Research). 39:e118-e118), or can be predicted using clinical data, e.g., ClinVar (Landrum et al. (2016) Nucleic Acids Research 44:D862-D868). Suitable additions, deletions, substitutions, modifications, exchanges, and / or variations may be considered acceptable, benign, and / or likely to be benign.

[0317] Typically, the variant may have some degree of identity with the wild-type amino acid sequence or wild-type nucleotide sequence.

[0318] In this context, a variant sequence is interpreted as containing an amino acid sequence that is 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 to the target sequence, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Variants may also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), but in the context of the present invention, it is preferable to express them in terms of sequence identity.

[0319] In this context, a variant sequence is interpreted as containing a nucleotide sequence that is 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 to the target sequence, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. While variants may also be considered in terms of similarity, in the context of the present invention, it is preferable to express them in terms of sequence identity.

[0320] Preferably, a reference to a sequence having percent identity with any of the sequence numbers described herein refers to a sequence having percent identity described over the entire length of the sequence number being referenced.

[0321] Sequence identity comparison can be performed visually or, more commonly, with the assistance of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage identity between two or more sequences.

[0322] Percent identity can be calculated over consecutive sequences; that is, one sequence is aligned with the other, and each amino acid or nucleotide in one sequence is directly compared, one residue at a time, with the corresponding amino acid or nucleotide in the other sequence. This is called a "gapless" alignment. Typically, such gapless alignments are performed only for a relatively short number of residues.

[0323] While this is a very simple and consistent method, it does not take into account, for example, that in otherwise identical sequence pairs, a single insertion or deletion in an amino acid or nucleotide sequence may cause subsequent residues or codons to fall out of alignment, and therefore, if a global alignment is performed, this could potentially result in a significant decrease in percentage identity. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes possible insertions and deletions into account without excessively penalizing the overall identity score. This is achieved by attempting to maximize local identity by inserting "gaps" into the sequence alignment.

[0324] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, so for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting a higher relevance between the two sequences being compared, will achieve a higher score than one with many gaps. A "affine gap cost" is typically used, which imposes a relatively high cost for the presence of gaps and a smaller penalty for each subsequent residue within the gap. This is the most commonly used gap scoring system. A high gap penalty results in an optimized alignment with fewer gaps. Most alignment programs allow for correction of the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

[0325] Therefore, calculating the maximum percentage identity requires first generating the optimal alignment, taking gap penalties into account. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (Devereux et al. (1984) Nucleic Acids Research 12:387-395). Examples of other software that can perform sequence comparisons include the BLAST package (Altschul et al. (1990) Journal of Molecular Biology 215:403-410), BLAST 2 (Tatusova et al. (1999) FEMS Microbiology Letters 174:247-250), FASTA (Pearson et al. (1988) PNAS 85:2444-2448), and EMBOSS Needle. Examples include, but are not limited to, Madeira et al. (2019) Nucleic Acids Research 47:W636-W641 and the GENEWORKS comparison tool suite. For some applications, it is preferable to use EMBOSS Needle.

[0326] While it is possible to measure the final percentage of identity, the alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​commonly used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix.

[0327] Once the software creates the optimal alignment, it is possible to calculate the percentage sequence identity. The software typically does this as part of a sequence comparison and generates a numerical result. Percent sequence identity can be calculated as the number of identical residues as a percentage of the total residues in the referenced sequence number. “Fragment” is also a variant, and this term typically refers to a selected region of the polypeptide or polynucleotide of interest, functionally or, for example, in an analytical method. Therefore, “fragment” refers to an amino acid sequence or nucleic acid sequence that is part of a full-length polypeptide or full-length polynucleotide.

[0328] Such variants, derivatives, and fragments can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. If an insertion is performed, a synthetic DNA encoding the insertion can be prepared, along with 5' and 3' facultative regions corresponding to the naturally occurring sequence on either side of the insertion site. Since the facultative regions contain convenient restriction sites corresponding to locations in the naturally occurring sequence, this sequence can be cleaved with a suitable enzyme(s), and the synthetic DNA can be ligated to these cleavage sites. The DNA is then expressed according to the present invention to produce an encoded polypeptide. These methods are merely examples of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0329] Those skilled in the art will understand that all features of the invention disclosed herein can be combined without departing from the scope of the invention as disclosed herein.

[0330] Preferred features and embodiments of the present invention are described below as non-limiting examples.

[0331] Unless otherwise specified, the implementation of this invention will utilize conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology. These are within the scope of the skills of those skilled in the art. Such techniques are described in the literature. For example, Sambrook, J., Fritsch, EF and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JMand McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DMand Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures. See Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference. [Examples]

[0332] Example 1 Materials and methods Plasmid construction Plasmids were constructed using a combination of standard cloning techniques and gene synthesis. The pMAX.coPCSK9 plasmid and the pMAX.coPCSK9.S127R plasmid were generated by replacing the GFP sequence in the plasmid pMAX.GFP(Amaxa) with the codon-optimized (co)gene-synthesized PCSK9 and PCSK9.S127R(GenScript), respectively.

[0333] Preparation of plasmid DNA Large-scale preparation of plasmid DNA was performed using the Macherey-Nagel endotoxin-free high-purity plasmid maxi prep system, following the manufacturer's instructions. After lysing cells and precipitating genomic DNA (gDNA) with a modified alkali / sodium dodecyl sulfate (SDS) procedure, the clear lysate was passed through an ion-exchange column at the appropriate salt concentration and pH. Plasmid DNA was eluted under high-salt conditions. It was then desalted and concentrated by alcohol precipitation. Finally, the plasmid DNA was resuspended in TE (10 mM TrisHCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA)).

[0334] LV production A pseudotyped third-generation self-inactivating LV vector, VSV.G, was produced by transient transfusion of calcium phosphate into 293T cells. 9 million 293T cells were seeded in 15 cm dishes 24 hours before transfusion. Two hours before transfusion, the culture medium was prepared. The dishes were replaced with 22.5 mL of fresh medium. For each dish, a solution containing a mixture of selected transfer plasmids, packaging plasmids pMDLg / pRRE and pCMV.REV, pMD2.G, and pAdVantage(Promega) plasmids was prepared using 35, 12.5, 6.25, 9, and 15 μg of plasmid DNA, respectively. To produce LV with PCSK9 overexpression, plasmids pMAX.coPCSK9 or pMAX.coPCSK9.S127 were added to the mixture using 7.5 and 3.75 μg of DNA, respectively. 0.1× TE solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0, distilled water) was added to the DNA mixture to a final volume of 1250 μL. The solutions were left on a rotating wheel for 20–30 minutes. During this time, 125 μL of CaCl2 was added to the mixture in equal portions into sampling tubes. Immediately before translocation, a precipitate was formed by adding 1250 μL of 2× HBS (281 mM NaCl, 100 mM HEPES, 1.5 mM Na₂HPO₄, pH 7.12) to the solution while stirring it over a vortex. The precipitate was immediately added to the culture medium and left on the cells for 14-16 hours. The culture medium was then replaced with 16 mL of fresh culture medium. 54 hours after the medium change, the supernatant containing the vector was collected. The supernatant was then passed through a 0.22 μm filter (Millipore). The filtered supernatant was either frozen at -80°C (unconcentrated LV) or transferred to a sterile 25 × 89 mm polyalomer tube (Beckman) and centrifuged at 20,000 g for 120 minutes at 20°C (Beckman Optima XL-100K ultracentrifuge). The vector pellet was dissolved in an appropriate volume of phosphate-buffered saline (PBS) to a concentration 500-fold.

[0335] Titration of LV and determination of vector copy number (VCN) For titration of LV, 1 × 10 5 293T cells were transduced using serial LV dilutions in the presence of polyblen (8 μg / mL) (1:10 for concentrated LV). 3 ~10 7For non-concentrated LV, 1:10 1 ~10 5For LV encoding PGK.GFP, cells were analyzed by flow cytometry 3–7 days after transduction, and the infectivity titer, expressed as transduction units (TU) / mL, was calculated using the formula TU / mL = (%GFP-positive cells / 100) × 100,000 × (1 / dilution factor). For all other LV, gDNA was extracted 10 days after transduction using the Maxwell 16 Cell DNA Purification Kit (Promega) according to the manufacturer's instructions. Vector copy number (VCN) was determined by digital droplet polymerase chain reaction (ddPCR) using primers (HIV fw: 5'-TACTGACGCTCTCGCACC-3'; HIV rv: 5'-TCTCGACGCAGGACTCG-3') and probes (FAM 5'-ATCTCTCTCCTTCTAGCCTC-3') designed for the primer binding site region of the LV, starting with 5–20 ng of template gDNA. The amount of endogenous DNA was quantified using primer / probe sets designed for the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (Applied Biosystems HS00483111_cm) or the TATA-box binding protein associated factor 7 (TAF7) gene (Applied Biosystems Rh02916247_s1). PCR reactions were performed using each primer (900 nM) and probe (250 nM) according to the manufacturer's instructions (Bio-Rad), read with a QX200 reader, and analyzed with QuantaSoft software (Bio-Rad). VCN was calculated using the formula = (ng LV / ng endogenous DNA) × 2.Each ddPCR run contained an internal standard generated by using a CEM cell line stably harboring six vector integrations, previously determined by Southern blotting and fluorescence in situ hybridization (FISH) analysis. Infectivity titer, expressed as TU / mL, was calculated using the formula TU / mL = VCN × 100,000 × (1 / dilution factor). LV physical particles were measured by HIV-1 Gag p24 antigen immunocapsulation analysis (Perkin Elmer) according to the manufacturer's instructions. LV-specific infectivity was calculated as the ratio between infectivity titer and physical particles.

[0336] For LV VCN determination in cell lines and T cells, DNA was extracted using the Maxwell 16 Cell DNA Pyrification Kit (Promega). VCNs were then quantified by ddPCR, as previously described for LV titration. For mouse experiments, DNA was extracted from whole liver samples using the Maxwell 16 Cell DNA Purification Kit (Promega). VCNs in mouse DNA were determined by ddPCR, starting with 5–20 ng of template gDNA, using primer / probe sets designed for the LV primer binding site region, as described for LV titration. The amount of endogenous mouse DNA was quantified using primer / probe sets designed for 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 using each primer (900 nM) and probe (250 nM) according to the manufacturer's instructions (Bio-Rad), read with a QX200 reader, and analyzed with QuantaSoft software (Bio-Rad).

[0337] cell culture 293T cells were maintained in Iskoff-modified Dulbecco's medium (IMDM, Sigma) supplemented with 10% fetal bovine serum (FBS, Euroclone), penicillin, and streptomycin 100 international units (IU) / mL (Lonza). Subconfluent cell lines were washed with sterile PBS (Sigma) and detached with 0.05% trypsin in PBS and 4 mM EDTA.

[0338] Primary human lymphocytes were magnetically sorted from the buffy coats of three healthy donors (Miltenyi Biotec). These were then administered to IMDM supplemented with interleukin-7 (5 ng / mL), interleukin-15 (5 ng / mL), 10% FBS, 1% glutamine, penicillin 100 IU / mL, and streptomycin 100 μg / mL. 6 Cells were cultured at a concentration of 10 cells / mL. T lymphocytes were pre-stimulated in vitro using beads coated with α-human-CD3 and α-human-CD28 (3 beads / cell, DynaBeads Human T-Activator CD3 / CD28, Invitrogen).

[0339] All cells were maintained at 37°C in a 5% CO2 humidified atmosphere.

[0340] Flow cytometry Flow cytometry analysis was performed using a FACSCanto analyzer (BD Biosciences) equipped with DIVA software. 100,000–500,000 cells were harvested and washed with PBS or MACS buffer (PBS pH 7.2, 0.5% bovine serum albumin, 2 mM EDTA). If the antibody was stained, it was treated with fragment crystallizable (Fc) Receptor-Block (Miltenyi Biotec, 1:100) and then resuspended in the buffer used for washing. LDLR staining was performed in 100 μL of MACS buffer, and the cells were cultured in the dark at 4°C for 20 minutes with antibody anti-LDLR conjugated to APC (clone 472413, dilution 1:10, R&D Systems).

[0341] In vitro experiments Transduction of cell lines and human primary cells was performed by adding the desired multiplicity of infection (MOI) of LV, which had been pre-counted, to the plated cells. The MOI was calculated as the number of vector TU per cell.

[0342] Enzyme-linked immunosorbent assay (ELISA) for PCSK9 The concentration of human PCSK9 in the LV-containing supernatant was determined by ELISA specific for the human PCSK9 antigen (Human Proprotein Convertase 9 / PCSK9 DuoSet ELISA, R&D Systems) according to the manufacturer's instructions. The absorbance of each sample was spectrophotometrically determined at 450 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific) and normalized against the standard curve.

[0343] Mouse experiments C57BL / 6 mice were purchased from Charles River Laboratories. All mice were maintained under specific pathogen-free conditions. Vector administration was performed by tail vein injection at a LV dose of 2.5×10 10 TU / Kg in adult C57BL / 6 mice. Blood was collected from the retro-orbital plexus of mice using a capillary and the blood was collected in 0.38% sodium citrate buffer (pH 7.4) for plasma preparation. All animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee.

[0344] ELISA for factor IX The concentration of human factor IX (FIX) in mouse plasma was determined by ELISA specific for the human FIX antigen (Asserachron IX:Ag, Stago) according to the manufacturer's instructions. Mouse plasma samples were diluted 1:50 or 1:100. The absorbance of each sample was spectrophotometrically determined at 450 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific) and normalized against the Ag standard curve.

[0345] Electron microscope Several microliters of concentrated LV batches were absorbed onto glow-discharge carbon-coated Formvar copper grids and fixed with 4% paraformaldehyde in PBS for 20 minutes. After several washes with 50 mM glycine in PBS, the grids were blocked with 1% BSA in PBS and cultured with primary antibody (anti-VSV.G, KeraFAST, 1:50) diluted in blocking buffer for 30–90 minutes. After several washes with 0.1% BSA in PBS, the samples were cultured with protein A-gold (5 or 10 nm) for 30 minutes, fixed with 1% glutaraldehyde, stained with 2% uranyl acetate or a mixture of 0.4% uranyl acetate and 1.8% methylcellulose, and then air-dried. The grids were observed with a Zeiss LEO512 transmission electron microscope. Images were acquired by a 2k×2k bottom-mounted slow-scanning ProScan camera controlled by EsivisionPro3.2 software. To quantify the label density, random images of virus particles were taken at a nominal magnification of 16k, and gold particles associated with virions were manually counted using ImageJ. Virians were defined based on their expected size (approximately 120nm) and high electron density cores.

[0346] result The inventors generated two plasmids encoding human wild-type PCSK9 or mutant (S127R) PCSK9 to obtain protein overexpression by adding one of these plasmids to the translocation mixture during LV production. The inventors produced LV expressing GFP under the control of a ubiquitous phosphoglycerate kinase (PGK) promoter (PGK.GFP), with or without the addition of the PCSK9-encoding plasmid. To increase the productivity and reinfection potential of producer cells, the inventors collected the LV-containing supernatant 54 hours after translocation and 1 day later compared to a standard protocol. In this setting, the inventors tested whether PCSK9 overexpression could lead to an increase in the former by decreasing the latter. Interestingly, the inventors observed 1.6-fold and 2-fold increases in infectivity titers upon addition of pMAX.coPCSK9 or pMAX.coPCSK9.S127R, respectively, to the translocation mixture (Figure 1A). Physical particles and infectivity were also slightly increased (Figures 1B and 1C). The inventors also measured the concentration of PCSK9 protein in LV-containing medium by ELISA and found a 2.9-fold decrease in protein when cells were transfused with a plasmid encoding mutant PCSK9 compared to the wild-type morphology (Figure 1D). Subsequently, after collecting the LVV-containing medium, the inventors performed flow cytometry analysis of producer cells to measure the LDLR content on their membranes. The inventors detected a significant decrease in the mean of fluorescence intensity (MFI) of LDLR when cells were transfused with pMAX.coPCSK9.S127R (Figure 1E), indicating effective inhibition of LDLR recycling on the plasma membrane of producer cells.

[0347] Next, the inventors decided to test the transduction ability of LV produced in the presence of PCSK9 by utilizing a clinically relevant model. First, the inventors transduced primary human T cells from three different donors in vitro with PGK.GFP LV produced with or without pMAX.coPCSK9.S127R. The inventors observed a 1.5 to 2-fold increase in VCN in cells transduced with PCSK9-produced LV compared to cells transduced with control LV (Figure 2A). Next, the inventors produced LV encoding human factor IX (hFIX) under the control of a hepatocyte-specific expression cassette (ET.FIX) in or without the PCSK9-encoding plasmid, and administered them intravenously to adult C57BL / 6 mice. Mouse plasma was collected and the amount of hFIX was quantified over time, and liver was collected 12 weeks after LV administration to measure VCN. The blood concentrations of hFIX and VCN in mice treated with LV produced by pMAX.coPCSK9.S127R were 1.5 to 2 times higher than in mice treated with control LV (Figures 2B and 2C). Mice treated with LV produced by pMAX.coPCSK9 showed comparable levels of hFIX and VCN to control mice. Overall, these data suggest that while overexpression of wild-type PCSK9 does not negatively affect the transduction efficiency of LV, LV produced in the presence of PCSK9.S127R may be more efficient than control LV in transduction of human T cells in vitro and mouse liver in vivo. We hypothesized that LDLR binds to VSV.G intracellularly during LV production, causing VSV.G sequesteration and thus reducing the VSV.G content on the LV envelope. In this scenario, overexpression of PCSK9.S127R, which induces LDLR degradation, may also affect the availability of VSV.G for incorporation into the LV envelope. Previous studies by our group have shown that a higher VSV.G content on LV particles corresponds to higher transduction efficiency, particularly in T cells and hematopoietic stem cells and hematopoietic progenitor cells.Therefore, the inventors quantified the amount of VSV.G on the envelope of LV particles produced with and without pMAX.coPCSK9.S127R by immunostaining and electron microscopy (Figures 3A and 3B). Surprisingly, the inventors observed a 1.5-fold increase in the amount of VSV.G on the envelope of virions produced in the presence of PCSK9 compared to control virions (Figure 3C). These data indicate that overexpression of the LDLR inhibitor PCSK9.S127R during LV production increases the LV infectivity titer by approximately twofold, resulting in improved LV production accompanied by increased VSV.G surface content and higher transduction efficiency for clinically relevant cell types such as T cells.

[0348] Various features and embodiments of the present invention will be described with reference to the following numbered paragraphs. 1. Enveloped virus particle producer cells or packaging cells, wherein the cells are modified to reduce the expression of low-density lipoprotein receptors (LDLRs) on the cell surface. 2. The cells are those described in paragraph 1, which contain heterogeneous polynucleotides including a nucleotide sequence encoding the proprotein convertase subtilisin / kexin type 9 (PCSK9). 3. A method for producing enveloped virus particles, (a) A step of introducing a transfer vector and one or more helper vectors into cells, (b) A step of introducing a vector containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into cells, (c) A method for producing enveloped virus particles, comprising the step of culturing cells under conditions suitable for the production of enveloped virus particles. 4. The nucleotide sequence encoding PCSK9 is: (a) A nucleotide sequence having at least 75% sequence identity with SEQ ID NO: 1 or 2, or consisting thereof, (b) The cell or method described in paragraph 2 or 3, comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 or 2. 5. PCSK9 is, (a) Containing or consisting of an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 3 or 4, (b) A cell or method according to any one of paragraphs 2 to 4, comprising or consisting of the amino acid sequence of Sequence ID No. 3 or 4. 6. The cells or method described in any one of paragraphs 2-5, wherein PCSK9 contains the mutation S127R, and the amino acids are numbered with reference to Sequence ID No. 4. 7. Cells are HEK-293 cells or derivatives thereof, as described in any one of paragraphs 1 to 6. 8. The enveloped virus particle is a lentivirus, as described in any one of paragraphs 1 to 7, in the cell or method described in any one of paragraphs 1 to 7. 9. The enveloped virus particle is a pseudotype of vesicular stomatitis virus glycoprotein G (VSV-G), as described in any one of paragraphs 1 to 8, in the cell or method described in any one of paragraphs 1 to 8. 10. Use of enveloped virus particle producer cells described in any one of paragraphs 1-2 or 4-9 for the production of enveloped virus particles. 11. A method for producing enveloped virus particles, (a) A step of providing an enveloped virus particle producer cell as described in any one of paragraphs 1-2 or 4-9, (b) A method for producing enveloped virus particles, comprising the step of culturing cells under conditions suitable for the production of enveloped virus particles. 12. Enveloped virus particles produced by the method described in any one of paragraphs 3-9 or 11. 13. Cells transduced by enveloped virus particles as described in paragraph 12. 14. Enveloped virus particles or cells as described in paragraph 12 or 13, for use in therapeutic purposes. 15. The treatment comprises administering enveloped virus particles to a subject in need thereof, as described in paragraph 14.

[0349] All publications referenced in the above specification are incorporated herein by reference. Various modifications and variations of the disclosed viral particles, cells, methods or uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention is disclosed in relation to certain preferred embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments. In fact, various modifications of the disclosed form for carrying out the invention, which will be apparent to those skilled in the art, are intended to fall within the scope of the following claims.

Claims

1. Enveloped virus particle producer cells or packaging cells, wherein the cells are modified to reduce the expression of low-density lipoprotein receptors (LDLRs) on the surface of the cells.

2. The cell according to claim 1, wherein the cell comprises heterogeneous polynucleotides including a nucleotide sequence encoding the proprotein convertase subtilisin / kexin type 9 (PCSK9).

3. A method for producing enveloped virus particles, (a) A step of introducing a transfer vector and one or more helper vectors into cells, (b) A step of introducing a vector containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into the cells, (c) A method for producing enveloped virus particles, comprising the step of culturing the cells under conditions suitable for the production of the enveloped virus particles.

4. A method for producing enveloped virus-like particles (VLPs), (a) A step of introducing one or more helper vectors into cells, (b) A step of introducing a vector containing a nucleotide sequence encoding proprotein convertase subtilisin / kexin type 9 (PCSK9) into the cells, (b) A method for producing enveloped virus-like particles (VLPs), comprising the step of culturing the cells under conditions suitable for the production of the enveloped VLPs.

5. The nucleotide sequence encoding PCSK9 is, (a) A nucleotide sequence having at least 75% sequence identity with SEQ ID NO: 1 or 2, or consisting of such a sequence, (b) A cell or method according to any one of claims 2 to 4, comprising or consisting of the nucleotide sequence of Sequence ID No. 1 or 2.

6. The aforementioned PCSK9 is (a) Containing or consisting of an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 3 or 4, (b) A cell or method according to any one of claims 2 to 5, comprising or consisting of the amino acid sequence of Sequence ID No. 3 or 4.

7. The cell or method according to any one of claims 2 to 6, wherein the PCSK9 comprises the mutation S127R, and the amino acids are numbered with reference to Sequence ID No.

4.

8. The cell or method according to any one of claims 1 to 7, wherein the cell is a HEK-293 cell or a derivative thereof.

9. The cell or method according to any one of claims 1 to 8, wherein the enveloped virus particle is a lentivirus, or the enveloped VLP is a lentivirus VLP.

10. The cell or method according to any one of claims 1 to 9, wherein the enveloped virus particle or enveloped VLP is a pseudotype of bullous stomatitis virus glycoprotein G (VSV-G).

11. Use of the enveloped virus particle producer cell according to any one of claims 1 to 2 or 5 to 10 for the production of the enveloped virus particles.

12. Use of the enveloped virus particle packaging cell according to any one of claims 1 to 2 or 5 to 10 for the production of enveloped virus-like particles (VLPs).

13. A method for producing enveloped virus particles, (a) A step of providing an enveloped virus particle producer cell according to any one of claims 1 to 2 or 5 to 10, (b) A method for producing enveloped virus particles, comprising the step of culturing the cells under conditions suitable for the production of the enveloped virus particles.

14. A method for producing enveloped virus-like particles (VLPs), (a) A step of providing an enveloped virus particle packaging cell according to any one of claims 1 to 2 or 5 to 10, (b) A method for producing enveloped virus-like particles (VLPs), comprising the step of culturing the cells under conditions suitable for the production of enveloped VLPs.

15. Enveloped virus particles produced by the method described in any one of claims 3, 5 to 10, or 13.

16. Enveloped virus-like particles (VLPs) produced by the method according to any one of claims 4 to 10 or 14.

17. Cells transduced by enveloped virus particles according to claim 15 or enveloped VLPs according to claim 16.

18. Enveloped virus particles, VLPs, or cells according to any one of claims 15 to 17, for use in therapeutic purposes.

19. The enveloped virus particle or VLP for use according to claim 18, wherein the treatment comprises administering the enveloped virus particle or VLP to a subject requiring it.

Images