WO2023170681A1 - Lentiviral vectors for in vivo targeting of immune cells - Google Patents

Lentiviral vectors for in vivo targeting of immune cells Download PDF

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WO2023170681A1
WO2023170681A1 PCT/IL2023/050234 IL2023050234W WO2023170681A1 WO 2023170681 A1 WO2023170681 A1 WO 2023170681A1 IL 2023050234 W IL2023050234 W IL 2023050234W WO 2023170681 A1 WO2023170681 A1 WO 2023170681A1
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acid sequence
vsv
nucleic acid
lentivirus
cell
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PCT/IL2023/050234
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Liron Elkouby
Noam DIAMANT
Einat NISSIM-ELIRAZ
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Noga Therapeutics Ltd.
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Definitions

  • the present invention is directed to lentiviral vectors optimized for in-vivo cell specific targeted transduction.
  • LVs Self-inactivating 3 rd generation lentiviral vectors
  • GT ex vivo gene therapy
  • HSC autologous hematopoietic stem cell
  • chimeric antigen receptor T cell therapy and others.
  • HSCs or T cells are isolated from the patient, transduced by LVs, and then readministered to the patient.
  • ex vivo genetic modification is complex and requires sophisticated facilities and skilled personnel, which makes this a high-cost personalized treatment.
  • ex vivo LV GT has proven to be safe and efficient with unparalleled ability to safely introduce large DNA segments to the genome of hematopoietic stem cells and T cells.
  • In-vivo LV GT by direct injection of the gene-correcting vector into the blood stream of a subject holds a great promise for many reasons. In-vivo administration of the vector omits the labor-intensive and costly ex vivo process.
  • In-vivo LV GT would reduce the loss of “sternness” of HSCs, therefore, will increase homing/engraftment capacity as there is no need to culture the target cells.
  • in-vivo LV GT has the potential to eliminate the need for conditioning, thus, dramatically decreasing side effects of the procedure. Accordingly, in-vivo LV GT will be more readily distributed to the patients, and potentially will even allow therapy re-administration as required.
  • Two main challenges for in vivo LV GT include: (1) lack of specific cell targeting; and (2) serum inactivation of the virus envelope glycoprotein mediated by the complement system.
  • VSV-G vesicular stomatitis virus G
  • VSV Vesicular stomatitis virus
  • G plays a critical role during the initial steps of virus infection. First, it is responsible for viral attachment to specific receptors. After binding, virions enter the cell by a Clathrin-mediated endocytic pathway. In the acidic environment of the endocytic vesicle, G triggers the fusion between the viral and endosomal membranes, which releases the viral genome into the cytosol, initiating the subsequent steps of infection. Fusion is catalyzed by a low-pH-induced large structural transition from a pre-fusion to a post-fusion conformation, which are both trimeric.
  • LDL-R Low-density lipoprotein receptor
  • LDL-R and its family members are not universal to all vesiculoviruses. While the close phylogenetic relatives to VSV such as Cocal, and Maraba viruses also interact with LDL-R, it has been shown that Piry and Chandipura viruses do not.
  • novel binding mutations of VSV-G demonstrating reduced binding capabilities without effecting the membrane fusion capacity.
  • we aligned and compared different vesiculoviruses family members both positive and negative LDL-R interactors. This comparison revealed conserved residues among the LDL-R binders family members which were not conserved among the family members utilizing other receptors for entry.
  • scFvs aimed at surface cell markers expressed on different hematopoietic cells were introduced on the membrane of the viral vector. Transduction of target cells is achieved in two steps: (i) Target cell binding via selected scFvs; and (ii) fusion to the endosome membrane by the non-binding VSV-G mutans. This enables the targeting of specific hematological cell populations such as HSCs, T or NK cells in vivo.
  • Serum inactivation - VSV-G serum inactivation was first described over 20 years ago.
  • One option to address this challenge is by replacement of the attachment glycoprotein with that of other viruses.
  • international patent application No. PCT/US2010/033616 teaches Cocal virus pseudotyped LVs with increased serum resistance.
  • CRPs complement regulatory proteins
  • DAF decay-accelerating factor
  • MCP membrane cofactor protein
  • the present invention discloses increasing serum resistance by optimizing the ratio of the envelope protein (e.g., VSV-G, cocal , RD114) and binding moieties (such as scFv against CD90 or CD34) on the viral membrane. Optimization of this ratio allows increasing serum resistance while maintaining high infectivity rates of a selected target cell population. Furthermore, the present invention, in some embodiments, is based, at least in part, on the surprising results showing that LV s expressing the novel reduced LDL receptor binding variants have increased resistance to human serum.
  • the envelope protein e.g., VSV-G, cocal , RD114
  • binding moieties such as scFv against CD90 or CD34
  • a method for preparing an improved lentivirus targeting a hematopoietic cell for in vivo gene therapy comprising: contacting a cell with: (a) a first expression vector comprising a nucleic acid sequence encoding a mutated vesicular stomatitis virus G protein (VSV-G), the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G; and (b) a second expression vector comprising a nucleic acid sequence comprising: (i) a nucleic acid sequence encoding an antibody heavy chain variable region (VH); (ii) a nucleic acid sequence encoding an antibody light chain variable region (VL); and (iii) a nucleic
  • a method for improving in vivo gene therapy targeting a hematopoietic cell in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a lentivirus comprising: (a) a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G; and (b) a second nucleic acid sequence comprising a sequence encoding an antibody VH; a sequence encoding an antibody VL; and a sequence encoding a transmembrane domain, wherein the encoded VH and VL form a scFv, wherein the lentivirus is obtained or derived from a cell comprising a first nucleic acid sequence encoding a
  • a synthetic polynucleotide comprising a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G.
  • an expression vector comprising the synthetic polynucleotide disclosed herein.
  • a viral vector comprising the expression vector disclosed herein.
  • a cell comprising: (a) the synthetic polynucleotide disclosed herein; (b) the expression vector disclosed herein; (c) the viral vector disclosed herein; (d) the lentivirus disclosed herein; or (e) any combination of (a) to (d).
  • composition comprising: (a) the synthetic polynucleotide disclosed herein; (b) the expression vector disclosed herein; (c) the viral vector disclosed herein; (d) the lentivirus disclosed herein; (e) the cell disclosed herein; or (f) any combination of (a) to (e); and an acceptable carrier.
  • the improved lentivirus is characterized by: reduced resistance onset, increased stability, reduced serum inactivation, increased titer, increased infectivity or any combination thereof, in a subject administered therewith, compared to a control lentivirus.
  • the improving comprises: reducing resistance of the subject to the lentivirus, increasing stability of the lentivirus in the subject, reducing inactivation of the lentivirus in serum of the subject, or any combination thereof, compared to a control lentivirus.
  • the administering is via a route selected from the group consisting of: intravenous, local, intraosseous infusion, intrathymical, intrathecal, and any combination thereof.
  • the mutated VSV-G further comprises at least one amino acid substitution of positions 47-50, compared to the wildtype VSV-G.
  • the mutated V SV -G comprises: (i) a substitution of lysine at position 50 (K50) to threonine; (ii) a substitution of threonine at position 352 (T352) to alanine; or (iii) both (i) and (ii).
  • the mutated VSV-G is characterized by having reduced or no binding affinity to a low-density lipoprotein (LDL) receptor, compared to a control VSV-G.
  • LDL low-density lipoprotein
  • the scFv is characterized by having specific binding affinity to any one of cluster of differentiation CD 34, CD90, and both.
  • the nucleic acid sequence encoding the antibody VH comprises the nucleic acid sequence set forth in SEQ ID Nos: 3, 33, 37, or 11.
  • the nucleic acid sequence encoding the antibody VL comprises the nucleic acid sequence set forth in SEQ ID Nos: 5, 34, 38, or 13.
  • the lentivirus comprises the nucleic acid sequence set forth in SEQ ID Nos: 6, 29, 3 l or 14.
  • the encoded VH comprises the amino acid sequence set forth in SEQ ID Nos: 7, 35, 39, or 9.
  • the encoded VL comprises the amino acid sequence set forth in SEQ ID Nos: 15, 36, 40 orl7.
  • the formed scFv comprises the amino acid sequence set forth in SEQ ID Nos: 10, 30, 32 or 18.
  • the encoded transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 19-22.
  • the subject is afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor thereof.
  • the synthetic polynucleotide further comprising a second nucleic acid sequence comprising: (a) a nucleic acid sequence encoding an antibody VH; (b) a nucleic acid sequence encoding an antibody VL; and (c) a nucleic acid sequence encoding a transmembrane domain, wherein the encoded VH and VL form a scFv.
  • the expression vector is a lentivirus-based expression vector.
  • the viral vector is a lentivirus.
  • the scFv is integrated into a viral envelope of the lentivirus via the transmembrane domain.
  • the carrier is a pharmaceutically acceptable carrier.
  • the composition is for use in in vivo gene therapy in a subject in need thereof.
  • Fig. 1 includes a non-limiting schematic description of a single-chain variable fragment (scFv) construct as disclosed herein.
  • Signal peptide, SP Variable heavy, VH; Linker; Variable light, VL; Transmembrane domain, TM; Cytoplasmatic tail, CT; and Linker, L.
  • Figs. 2A-2B include vertical bar graphs.
  • (2A) A vertical bar graph showing CD90 scFv expression on 293T cell membrane.
  • Each of the 8 different plasmid constructs containing the CD90 scFv fused to a different signal peptide and a transmembrane domain were co transfected with a GFP plasmid into 293T cells. Twenty-four hours post transfection cells were harvested and the scFv expression on the cell membrane was measured by flow cytometry using an anti-Myc antibody. Transfection efficiency was even in all tested constructs as determined by GFP expression (data not shown).
  • Fig. 3 includes a multiple sequence alignment of VSV (VSIVA), Cocal (COCAV), Maraba (MARAV), Piry (PIRYV) and Chandipura (CHAV) glycoproteins.
  • the consensus sequence is highlighted in black bold.
  • Matched amino acid are marked by a gray background.
  • CR2-CR3 binding segments marked with black rectangles.
  • Figs. 4A-4C include vertical bar graphs showing defected mutants infection capacity in comparison to wt VSV-G in different cells. Fold change infectivity in (4A) 293T, (4B) U937, and (4C) human primary CD34+ and activated T cells post transduction with WT or mutants VSV-G.
  • Fig. 5 includes plots of flow cytometry (FACS) analysis showing VSV-G mutants (K50T, and T352A) expression on 293T cells membrane in comparison to wt VSV-G.
  • FACS flow cytometry
  • Fig. 6 includes a vertical bar graph showing percentage of double positive VSV-G and CD90 scFv expression.
  • 293T cells were transfected with VSV-G (100 ng/pl) and CD90 scFv (100, 50 and 25 ng/pl). Twenty-four hours post transfection cells were harvested and VSV-G and CD90 scFv expression on cell membrane was measured by flow cytometry using anti-VSV-G and anti- Myc antibodies, respectively.
  • Figs. 7A-7C includes flow cytometry plots characterizing (7A) 293T_CD90+; (7B) 293T_CD34+; and (7C) 293T_MPL+ stable cell lines. After puromycin selection, the stable cell lines were incubated with antibodies against CD90, CD34 and MPL respectively. Flow cytometry was used to demonstrate successful markers expression.
  • Figs. 8A-8E includes vertical bar graphs and flow cytometry plots showing infection capacity of scFv with VSV-G K50T T352A i n comparison to VSV-G WT in 293T ⁇ marker and Jurkat cells. Cells were transduced with the different LVs and 3 days post transduction, and infection rates were measured.
  • (8A) A vertical bar graph showing scFv 90 _VSV-G K50T T352A i n comparison to VSV-G WT in 293T ⁇ CD90.
  • (8B) A vertical bar graph showing scFv 34 _VSV-G K50T - T352A i n comparison to VSV-G WT in 293T ⁇ CD34.
  • Figs. 9A-9C includes vertical bar graphs and flow cytometry plots showing infection capacity of VSV-G WT , scFv4C8_VSV-G K50T - T352A an d scFvCD3 VSV-G K50T - T352A i n primary HSC gated based on CD34 levels (high and low), activating and resting T cells. Cells were transduced with the different LVs and 4 days post transduction; infection rates were measured. (9A) A flow cytometry plot sowing CD34 cells gating strategy.
  • Figs. 10A-10B include vertical bar graph showing infection capacity of VSV-G WT and scFv4C8_VSV-G K50T - T352A in (10A) hepatocytes cell lines and (10B) florescent microscopy images in mouse primary hepatocytes.
  • Figs. 11A-11C include vertical bar graphs and flow cytometry plots.
  • Fig. 12 includes a graph showing that the infectivity level of WT VSV-G is more sensitive to reduction in VSV-G levels compared to T352A mut VSV-G.
  • Fig. 13 includes a vertical bar graph showing that the VSV-G WT is more sensitive to human serum compared to scFvVSV-GT352A.
  • a method for preparing an improved lentivirus targeting a hematopoietic cell for in vivo gene therapy is provided.
  • the method comprises contacting a cell with: (a) a first expression vector comprising a nucleic acid sequence encoding a mutated vesicular stomatitis virus G protein (VSV-G), the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) positions 47-50; (iii) positions 184-189; (iv) positions 349-353; and (v) any combination of (i) to (iv), compared to a wildtype VSV-G; and (b) a second expression vector comprising a nucleic acid sequence comprising: (i) a nucleic acid sequence encoding an antibody heavy chain variable region (VH); (ii) a nucleic acid sequence encoding an antibody light chain variable region (VL); and (iii) a nucleic acid sequence encoding a transmembrane domain, wherein the encoded VH and VL form a single expression vector comprising a nucle
  • the improved lentivirus is characterized by: reduced resistance onset, increased stability, reduced serum inactivation, improved titer, improved infectivity, or any combination thereof, in a subject administered therewith, compared to a control lentivirus.
  • the method comprises administering to a subject a therapeutically effective amount of a lentivirus comprising: (a) a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) positions 47-50; (iii) positions 184-189; (iv) positions 349-353; and (v) any combination of (i) to (iv), compared to a wildtype VSV-G; and (b) a second nucleic acid sequence comprising a sequence encoding an antibody VH; a sequence encoding an antibody VL; and a sequence encoding a transmembrane domain, wherein the encoded VH and VL form a scFv, wherein the lentivirus is obtained or derived from a cell comprising the first nucleic acid sequence and the second nucleic acid sequence at a copy number ratio of ranging
  • improving comprises: reducing resistance of the subject to the lentivirus, increasing stability and/or abundance of the lentivirus in the subject, reducing inactivation (such as by proteolytic enzyme(s) and/or protein(s), such as complement system proteins, but not limited thereto) of the lentivirus in serum of the subject, or any combination thereof, compared to a control lentivirus.
  • the contacting is at a copy number ratio ranging between: 0.01 : 1 and 1:1, 0.1:1 and 1:1, 0.05:1 and 1:1, 0.2:1 and 1:1, 0.3:1 and 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.01:0.99 and 0.1:0.9 of the first expression vector and the second expression vector.
  • a copy number ratio ranging between: 0.01 : 1 and 1:1, 0.1:1 and 1:1, 0.05:1 and 1:1, 0.2:1 and 1:1, 0.3:1 and 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.01:0.99 and 0.1:0.9 of the first expression vector and the second expression vector.
  • the first and second nucleic acid sequences as disclosed herein are located on a single expression vector. In some embodiments, the first and second nucleic acid sequences as disclosed herein, are located on separate expression vectors. In some embodiments, the first and second nucleic acid sequences as disclosed herein, are located on separate expression vectors, being the first and second expression vectors disclosed herein.
  • a synthetic polynucleotide comprising a nucleic acid sequence encoding a mutated vesicular stomatitis virus G (VSV-G) protein.
  • VSV-G mutated vesicular stomatitis virus G
  • the mutated VSV-G protein is characterized by having reduced or no binding affinity to a low-density lipoprotein (LDL) receptor.
  • LDL low-density lipoprotein
  • a synthetic polynucleotide comprising a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) positions 47-50; (iii) positions 184- 189; (iv) positions 349-353; and (v) any combination of (i) to (iv), compared to a wildtype VSV- G.
  • the mutated VSV-G further comprises at least one amino acid substitution of positions 47-50, compared to a wildtype VSV-G.
  • the synthetic polynucleotide further comprises a second nucleic acid sequence comprising: a nucleic acid sequence encoding an antibody heavy chain variable region (VH); a nucleic acid sequence encoding an antibody light chain variable region (VL); and a nucleic acid sequence encoding a transmembrane domain.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • the scFv is characterized by having specific binding affinity to any one of cluster of differentiation CD34, CD90, or both. In some embodiments, the scFv binds specifically to CD34, CD90, or both. In some embodiments, the scFv binds to CD34, CD90, or both, of a hematopoietic cell. In some embodiments, the scFv binds to CD34, CD90, or both, on the surface of a hematopoietic cell.
  • the antibody VH is encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID Nos: 3, 33, 37, or 11.
  • the antibody VL is encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID Nos: 5, 34, 38, or 13.
  • the synthetic polynucleotide further comprises a nucleic acid sequence encoding a linker being located between the nucleic acid sequence encoding the antibody VH and the nucleic acid sequence encoding the antibody VL.
  • the linker is encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 12 .
  • the synthetic polynucleotide comprises the nucleic acid sequence set forth in SEQ ID Nos: 6, 29, 31 or 14.
  • the encoded VH comprises the amino acid sequence set forth in SEQ ID Nos: 7, 35, 39, or 9.
  • the encoded VL comprises the amino acid sequence set forth in SEQ ID Nos: 15, 36, 40 orl7.
  • the encoded linker comprises the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 16.
  • the formed scFv comprises the amino acid sequence set forth in SEQ ID Nos: 10, 30, 32 or 18.
  • the encoded transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 19-22.
  • the synthetic polynucleotide further comprises a nucleic acid sequence encoding a signal or a leading peptide.
  • the encoded signal or leading peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the synthetic polynucleotide further comprises a promoter sequence, wherein any of the nucleic acid sequence encoding the antibody VH; the nucleic acid sequence encoding the antibody VL; and the nucleic acid sequence encoding the transmembrane domain is operably linked to the promoter sequence.
  • the synthetic polynucleotide further comprises at least one additional nucleic acid sequence encoding: a protein translation initiation motif, a tag, a cytoplasmic tail, a transcription termination motif, or any combination thereof.
  • the mutated VSV-G comprises a signal peptide. In some embodiments, the mutated VSV-G is devoid of a signal peptide.
  • the mutated VSV-G when being devoid of a signal peptide, comprises at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G.
  • the mutated VSV-G when being devoid of a signal peptide, further comprises at least one amino acid substitution in positions 47-50, compared to a wildtype VSV-G.
  • the mutated VSV-G when comprising a signal peptide, comprises at least one amino acid substitution in any one of: (i) positions 24-26; (ii) 200-205; (iii) 365-369; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G.
  • the mutated VSV-G when comprising a signal peptide, further comprises at least one amino acid substitution in positions 63-66, compared to a wildtype VSV-G. [091] In some embodiments, when being devoid of a signal peptide, the mutated VSV-G comprises a substitution of glutamic acid at position 352 (T352).
  • the mutated VSV-G when being devoid of a signal peptide, comprises a substitution of glutamic acid at position 352 (T352); and optionally a substitution of lysine at position 50 (K50), compared to a wildtype VSV-G.
  • the mutated VSV-G when being devoid of a signal peptide, comprises: (i) a substitution of glutamic acid at position 352 (T352) and (ii) a substitution of lysine at position 50 (K50), compared to a wildtype VSV-G.
  • the mutated VSV-G when being devoid of a signal peptide, comprises: (i) a substitution of lysine at position 50 (K50) to threonine (K50T); (ii) a substitution of threonine at position 352 (T352) to alanine (T352A); or (iii) both (i) and (ii), compared to a wildtype VSV-G.
  • the mutated VSV-G when comprising a signal peptide, comprises a substitution of threonine at position 368 (T368).
  • the mutated VSV-G when comprising a signal peptide, comprises a substitution of threonine at position 368 (T368), and optionally a substitution of lysine at position 66 (K66), compared to a wildtype VSV-G.
  • the mutated VSV-G when comprising a signal peptide, comprises: (i) a substitution of threonine at position 368 (T368); and (ii) a substitution of lysine at position 66 (K66), compared to a wildtype VSV-G.
  • the mutated VSV-G when comprising a signal peptide, comprises: (i) a substitution of lysine at position 66 (K66) to threonine (K66T); (ii) a substitution of threonine at position 368 (T368) to alanine (T368A); or (iii) both (i) and (ii), compared to a wildtype VSV- G.
  • wildtype VSV-G protein comprises the amino acid sequence: MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQV KMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQG TWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTV HNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACK MQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERIL DYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSR MVGMISGTTTEREEWDDWAPYEDVEIGPNGVERTSSGYKFPEY
  • wildtype VSV-G protein is encoded by the nucleic acid sequence: ATGAAGTGTCTGCTGTACCTGGCGTTCCTGTTTATCGGGGTGAACTGCAAGTTCACTA TCGTGTTTCCGCACAACCAAAAGGGCAACTGGAAAAACGTGCCTTCAAATTACCATT ATTGCCCCAGCAGCTCGGACCTGAACTGGCACAATGACCTCATTGGAACCGCGCTGC AGGTGAAGATGCCAAAGAGCCACAAGGCTATCCAGGCTGACGGATGGATGCCAC GCGTCAAAATGGGTGACTACCTGCGATTTCCGCTGGTACGGACCAAAATACATCACG CACAGCATCAGATCATTCACCCCGTCAGTGGAACAATGCAAAGAATCCATCGAACAG ACTAAGCAGGGAACCTGGCTGAACCCTGGATTTCCGCCGCAGTCGTGTGGGTACGCACGTGCTTGTG GAACAATGCAAAGAATCCATCGAACAG ACTAAGCAGGGAACCTGGCTGAACCCTGGATTTCCGCCGCAGTCGT
  • the term "functional analog” encompasses any analog of VSV-G protein having the same or equivalent activity attributed to a VSV-G protein and having an amino acid sequence differing from the wildtype VSV-G protein by at least one amino acid.
  • the synthetic polynucleotide further comprises a second nucleic acid comprising: (a) a first nucleic acid sequence encoding an antibody heavy chain variable region (VH); (b) a second nucleic acid sequence encoding an antibody light chain variable region (VL); and (c) a third nucleic acid sequence encoding a transmembrane domain.
  • the encoded VH and VL form a single-chain variable fragment (scFv).
  • a polynucleotide comprising: (a) a first nucleic acid sequence encoding an antibody heavy chain variable region (VH); (b) a second nucleic acid sequence encoding an antibody light chain variable region (VL); and (c) a third nucleic acid sequence encoding a transmembrane domain.
  • the encoded VH and VL form a single-chain variable fragment (scFv) having specific binding affinity to cluster of differentiation CD34 or CD90.
  • scFv single-chain variable fragment
  • the polynucleotide disclosed herein is an isolated polynucleotide.
  • the polynucleotide is a DNA molecule.
  • the DNA molecule is an isolated DNA molecule.
  • the polynucleotide or the DNA molecule is a complementary DNA (cDNA) molecule.
  • the first nucleic acid sequence comprises the nucleic acid sequence: CAAGTGAAGCTGCAAGAGTCTGGCCCTGGACTGGTGCAGCCTAGCCAGAGCCTGAG CTTCATCTGTACCGTGTCCGGCTTCAGCCTGACATCTCATGGCGTGCACTGGGTCCGA CAGAGCCCTGGAAAAGGACTGCAGTGGCTGGGAGTGATTTGGGGAGCCGGCAGAAC CGATTACAACGCCGCCTTCATCAGCAGACTGAGCATCAGCCGGGACATCAGCAAGA GCCAGGTGTTCTTCAAGATGAACAGCCTGCAGGTCGACGACACCGCCATCTACTACT GCGCCCGGAACAGATACGAGAGCTACTTCGACTATTGGGGCCAGGGCACCACCGTG ACAGTTTCTAGC (SEQ ID NO: 3).
  • the first nucleic acid sequence comprises the nucleic acid sequence:
  • the first nucleic acid sequence comprises the nucleic acid sequence:
  • the first nucleic acid sequence comprises the nucleic acid sequence: ATGGCTGTTCTGGGCCTGCTGCTGTGCCTGGTCACCTTTCCAAGCTGTGTGCTGAGCC
  • the first nucleic acid sequence comprises the nucleic acid sequence set forth in SEQ ID NOs: 3, 11, 33, or 37.
  • the second nucleic acid sequence comprises the nucleic acid sequence:
  • the second nucleic acid sequence comprises the nucleic acid sequence:
  • the second nucleic acid sequence comprises the nucleic acid sequence:
  • the second nucleic acid sequence comprises the nucleic acid sequence:
  • the second nucleic acid sequence comprises the nucleic acid sequence set forth in SEQ ID NOs: 5, 13, 34 or 38.
  • any one of the first and second polynucleotides further comprises a nucleic acid sequence encoding a linker.
  • the nucleic acid sequence encoding a linker is located between the first nucleic acid sequence, and the second nucleic acid sequence of the second polynucleotide, of the polypeptide disclosed herein.
  • the linker is a peptide or a proteinaceous linker. In some embodiments, the linker is a flexible linker.
  • sequence nucleic acid sequence encoding a linker comprises the nucleic acid sequence:
  • the nucleic acid sequence encoding a linker comprises the nucleic acid sequence:
  • the nucleic acid sequence encoding a linker comprises the nucleic acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 12.
  • the second polynucleotide comprises the nucleic acid sequence: CAAGTGAAGCTGCAAGAGTCTGGCCCTGGACTGGTGCAGCCTAGCCAGAGCCTGAG CTTCATCTGTACCGTGTCCGGCTTCAGCCTGACATCTCATGGCGTGCACTGGGTCCGA CAGAGCCCTGGAAAAGGACTGCAGTGGCTGGGAGTGATTTGGGGAGCCGGCAGAAC CGATTACAACGCCGCCTTCATCAGCAGACTGAGCATCAGCCGGGACATCAGCAAGA GCCAGGTGTTCTTCAAGATGAACAGCCTGCAGGTCGACGACACCGCCATCTACTACT GCGCCCGGAACAGATACGAGAGCTACTTCGACTATTGGGGCCAGGGCACCACCGTG ACAGTTTCTAGCGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTC TGATGCTGATGACCAACACACCCCTGAGCCTGCCTGTCTCTGGGAGATCAGGC CAGCATCAGCTGC
  • the second polynucleotide comprises the nucleic acid sequence:
  • the second polynucleotide comprises the nucleic acid sequence: GAGATTCAGCTGCAGCAGTCTGGCCCCGAGCTTATGAAGCCTGGCGCCTCTGTGAAG
  • the second polynucleotide comprises the nucleic acid sequence: ATGGCTGTTCTGGGCCTGCTGCTGTGCCTGGTCACCTTTCCAAGCTGTGTGCTGAGCC
  • the second polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NOs: 6, 14, 29 or 31.
  • the encoded VH comprises the amino acid sequence: QVKLQESGPGLVQPSQSLSFICTVSGFSLTSHGVHWVRQSPGKGLQWLGVIWGAGRTDY NAAFISRLSISRDISKSQVFFKMNSLQVDDTAIYYCARNRYESYFDYWGQGTTVTVSS (SEQ ID NO: 7).
  • the encoded VH comprises the amino acid sequence: DVLMTNTPLSLPVSLGDQASISCRSSQNLVHSNGNTYLHWYLQKPGQSPNLLIYKVSNRF SGVPDRFSGSGSGTEFTLKISRVQAEDLGVYFCSQSTHVPLTFGAGSKLELK (SEQ ID NO: 9).
  • the encoded VH comprises the amino acid sequence: EIQLQQSGPELMKPGASVKISCKASGYSFTSYYMHWVKQSQGKSLEWIGFIDPFNGGITY NQKFKGKATLTVDRSSSTAYMHLRSLTSEDSAVYYCARCYYNYDDEGRAMDYWGQGT SVTVSS (SEQ ID NO: 35).
  • the encoded VH comprises the amino acid sequence: MAVLGLLLCLVTFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVTWIRQPPG KGLEWLGVIWGGGSTYYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAKHES YYPFVYWGQGTLVTVSA (SEQ ID NO: 39).
  • the encoded VH comprises the amino acid sequence set forth in SEQ ID Nos: 7, 9, 35 or 39.
  • the encoded VL comprises the amino acid sequence:
  • the encoded VL comprises the amino acid sequence: DIRLTQSPSSLSASIGDRVTITCRASQGISRSLVWYQQKPGKAPRLLIYAASTLQSGVPSRF SGSGSGDFTLTISSLQPEDFATYYCLQHNTYPFTFGPGTKVDIKSGIPEQKL (SEQ ID NO: 17).
  • the encoded VL comprises the amino acid sequence: DIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQGPDGTFKRLIYATSSLDSSVPKR FSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPYTFGGGTKLEIKR (SEQ ID NO: 36).
  • the encoded VL comprises the amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLINSNGNTYLHWYLQKPGQSPKLLIHRVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKR (SEQ ID NO: 40).
  • the encoded VL comprises the amino acid sequence set forth in SEQ ID Nos: 14, 17, 36 or 40.
  • the linker or the encoded linker comprises the amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 8).
  • the linker or the encoded linker comprises the amino acid sequence: GSGGGGSGGGGSGGGGS (SEQ ID NO: 16).
  • the linker or the encoded linker comprises the amino acid sequence: SGGGGSGGGGSGGGGS (SEQ ID NO: 42).
  • the linker or the encoded linker comprises the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 16.
  • the formed scFv comprises the amino acid sequence: QVKLQESGPGLVQPSQSLSFICTVSGFSLTSHGVHWVRQSPGKGLQWLGVIWGAGRTDY NAAFISRLSISRDISKSQVFFKMNSLQVDDTAIYYCARNRYESYFDYWGQGTTVTVSSGG GGSGGGGSGGGGSDVLMTNTPLSLPVSLGDQASISCRSSQNLVHSNGNTYLHWYLQKPG QSPNLLIYKVSNRFSGVPDRFSGSGSGTEFTLKISRVQAEDLGVYFCSQSTHVPLTFGAGS KLELK (SEQ ID NO: 10).
  • the formed scFv comprises the amino acid sequence:
  • the formed scFv comprises the amino acid sequence: EIQLQQSGPELMKPGASVKISCKASGYSFTSYYMHWVKQSQGKSLEWIGFIDPFNGGITY NQKFKGKATLTVDRSSSTAYMHLRSLTSEDSAVYYCARCYYNYDDEGRAMDYWGQGT SVTVSSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQQ GPDGTFKRLIYATSSLDSSVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPYTFGG GTKLEIKR (SEQ ID NO: 30).
  • the formed scFv comprises the amino acid sequence: MAVLGLLLCLVTFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVTWIRQPPG KGLEWLGVIWGGGSTYYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAKHES YYPFVYWGQGTLVTVSASGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSS QSLINSNGNTYLHWYLQKPGQSPKLLIHRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED LGVYFCSQSTHVPWTFGGGTKLEIKR (SEQ ID NO: 32).
  • the formed scFv comprises the amino acid sequence set forth in SEQ ID NO: 10, 18, 30, or 32.
  • the encoded transmembrane domain comprises the amino acid sequence:
  • the encoded transmembrane domain comprises the amino acid sequence: AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 20).
  • the encoded transmembrane domain comprises the amino acid sequence:
  • the encoded transmembrane domain comprises the amino acid sequence: TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN RRRV (SEQ ID NO: 22).
  • the encoded transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 19-22.
  • any one of the first and second polynucleotides further comprises a nucleic acid sequence encoding a signal or a leading peptide.
  • the encoded signal or leading peptide comprises the amino acid sequence: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 1).
  • the encoded signal or leading peptide comprises the amino acid sequence: MKCLLYLAFLFIGVNCK (SEQ ID NO: 2).
  • the encoded signal or leading peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • any one of the first and second polynucleotides further comprises a promoter sequence.
  • any one of the first to third nucleic acid sequences is operably linked to the promoter sequence.
  • the polynucleotide further comprises at least one nucleic acid sequence encoding: a protein translation initiation motif, a tag, a cytoplasmic tail, a transcription termination motif, or any combination thereof.
  • a protein translation initiation motif comprises a mammalian, e.g., a human, protein translation initiation motif.
  • a protein translation initiation motif comprises a Kozak consensus sequence or any functional equivalent thereof.
  • an expression vector comprising the polynucleotide(s) disclosed herein. In some embodiments, there is provided an expression vector comprising the first polynucleotide and the second polynucleotide. In some embodiments, the expression vector comprises a plasmid. [0160] In some embodiments, there is provided a viral vector. In some embodiments, the viral vector comprises a lentiviral vector. In some embodiments, the viral vector is expressed, at least partially, from the expression vector disclosed herein. In some embodiments, the lentiviral vector is expressed, at least in part, from expression vector disclosed herein. In some embodiments, the expression vector is or comprises a lentivirus-based expression vector.
  • a lentivirus comprising a mutated VSV- G protein as disclosed herein.
  • a lentivirus comprising an antibody or a fragment thereof having specific binding affinity to a hematopoietic cell surface marker.
  • a lentivirus comprising a mutated VSV- G protein as disclosed herein, and an antibody or a fragment thereof having specific binding affinity to a hematopoietic cell surface marker.
  • a hematopoietic cell is a HSPC a myeloid cell, a lymphoid cell, an erythrocyte, a mast cell, a myeloblast, T cell, B cell, NK cell, macrophage, monocyte, dendritic cell, neutrophil, or an eosinophil.
  • the antibody or a fragment thereof is a scFv.
  • a lentivirus comprising a scFv having specific binding affinity to a HSPC cell surface marker.
  • HSPC surface markers are CD34, CD90, CD117, CD133, CD59, and Flt3.
  • a lentivirus comprising a scFv having specific binding affinity to a T cell surface marker.
  • T surface markers are CD3, CD2, CD4, CD5, CD7, CD8, CD16, CD16a, CD28, CCR5, and CD107b.
  • a lentivirus comprising a scFv having specific binding affinity to a NK cell surface marker.
  • NK surface markers are CD56, CD244 and CD16.
  • a lentivirus comprising a scFv having specific binding affinity to a macrophage cell surface marker.
  • useful macrophage surface markers are CD14, CD16, CD64, CD 68 and CDl lb.
  • a lentivirus comprising a scFv having specific binding affinity to CD34.
  • a lentivirus comprising a scFv having specific binding affinity to CD90 (also known as Thyl).
  • a lentivirus comprising: (i) a scFv having specific binding affinity to CD34; and (ii) a scFv having specific binding affinity to CD90.
  • a lentivirus comprising a bi-specific scFv having specific binding affinity to both CD34 and CD90.
  • any one of the scFvs is integrated into the viral envelope of the lentivirus disclosed herein. In some embodiments, any one of the scFvs is integrated into the viral envelope via a transmembrane domain. In some embodiments, any one of the scFvs is integrated into the viral envelope via a transmembrane domain as disclosed herein.
  • the lentivirus disclosed herein comprises a vesicular stomatitis virus G (VSV-G) protein. In some embodiments, the lentivirus disclosed herein comprises a viral envelope comprising a VSV-G protein.
  • VSV-G vesicular stomatitis virus G
  • a VSV-G protein is or comprises a mutated VSV-G protein.
  • a mutated VSV-G is characterized by having reduced or no binding affinity to a low-density lipoprotein (LDL) receptor.
  • LDL low-density lipoprotein
  • a mutated VSV-G protein maintains or preserves a pH-dependent membrane fusion.
  • a mutated VSV-G protein comprises any VSV-G protein comprising at least one amino acid substitution, deletion, insertion, substitution or incorporation of non- naturally occurring amino acid, compared to the wildtype VSV-G protein, as long as the mutated VSV-G protein is: (i) characterized by having reduced or no binding affinity to a LDL receptor; (ii) maintains or preserves a pH-dependent membrane fusion; or (iii) both (i) and (ii).
  • a control VSV-G protein comprises a wildtype VSV-G protein.
  • a control VSV-G protein comprises any mutated VSV-G protein comprising mutation(s) which do not affect and/or reduce and/or inhibit LDL receptor biding.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) and an antibody or a fragment thereof having specific binding affinity to a hematopoietic cell surface marker.
  • the antibody or a fragment thereof is a scFv.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD34
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD90.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD3, CD4, CD8 or any combination thereof.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) a scFv having specific binding affinity to CD34; and (iii) a scFv having specific binding affinity to CD90.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) a scFv having specific binding affinity to CD3; and (iii) a scFv having specific binding affinity to CD4.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) a scFv having specific binding affinity to CD3; and (iii) a scFv having specific binding affinity to CD8.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) a bi-specific scFv having specific binding affinity to both CD34 and CD90.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD3.
  • a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD56
  • an antibody refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen.
  • An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light” and one "heavy” chain. The variable regions of each light/heavy chain pair form an antibody binding site.
  • An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelid, CDR- grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitopebinding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences.
  • An antibody may be from any species.
  • the term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulfide-linked variable region (dsFv).
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof.
  • Fc region or fragment thereof an immunoglobulin domain including but not limited to, an Fc region or fragment thereof.
  • fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH) ⁇ Fc fusions and scFv-scFv-Fc fusions.
  • a lentivirus-based vector comprises an inactivated lentiviral vector.
  • a lentivirus-based vector comprises a third-generation lentiviral vector.
  • a lentivirus-based vector comprises a self-inactivating lentiviral vector.
  • a lentivirus-based vector is devoid of a trans-activator of transcription encoding gene (Tat).
  • a lentivirus-based vector is devoid of one or more viral accessory proteins.
  • a viral accessory protein is selected from: vif, vpr, vpu, nef, or any combination thereof.
  • a lentivirus-based vector comprises any lentivirus-based vector suitable for human therapy. In some embodiments, suitable comprises safe for human health.
  • a cell comprising: (a) any one of the polynucleotides disclosed herein; (b) the expression vector disclosed herein; (c) the lentivirus disclosed herein; or (d) any combination of (a) to (c).
  • the cell is a transduced cell.
  • transduced comprises being transduced with the expression vector of the invention.
  • the cell is an animal cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell or a human cell line.
  • the cell is configured to or optimized for expression, production, packing, secretion, assembly, or any combination thereof, of the lentivirus disclosed herein. [0199] In some embodiments, the cell is suitable for expression, production, packing, secretion, assembly, or any combination thereof, of the lentivirus disclosed herein.
  • the cell is used in a method for expression, production, packing, secretion, assembly, or any combination thereof, of the lentivirus disclosed herein.
  • the method comprises culturing the cell as disclosed herein under conditions sufficient for the cell to express the expression vector disclosed herein, thereby expressing, producing, packing, secreting, assembling, or any combination thereof, of the lentivirus disclosed herein.
  • composition comprising: (a) the polynucleotides disclosed herein; (b) the expression vector disclosed herein; (c) the lentivirus disclosed herein; (d) the cell disclosed herein, or (e) any combination of (a) to (d).
  • the composition further comprises an acceptable carrier, adjuvant, or excipient.
  • the carrier, adjuvant, or excipient is a pharmaceutically acceptable carrier, adjuvant, or excipient.
  • the composition is for use in in-vivo gene therapy in a subject in need thereof.
  • the subject is afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor cell thereof.
  • the composition is for use in transfection or transduction, or introduction of an exogenous nucleic acid molecule into a subject in need thereof.
  • in-vivo gene therapy comprises transfection or transduction, or introduction of an exogenous nucleic acid molecule into a subject in need thereof.
  • a method for preparing an improved lentivirus targeting a hematopoietic cell for in vivo gene therapy comprising: contacting a cell with: (a) a first expression vector comprising a nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G; and (b) a second expression vector comprising a nucleic acid sequence comprising: a nucleic acid sequence encoding an antibody heavy chain variable region (VH); a nucleic acid sequence encoding an antibody light chain variable region (VL); and a nucleic acid sequence encoding a transmembrane domain, wherein the encoded VH and VL form a
  • the contacting is at a copy number wherein the copy number of the second expression vector is equal to or greater than the copy number of the first expression vector. In some embodiments, the copy number of the second expression vector supersedes the copy number of the first expression vector. In some embodiments, the copy number of the second expression vector is greater than the copy number of the first expression vector. In some embodiments, the copy number of the first expression vector is lower than or equal to the copy number of the second expression vector.
  • the contacting is at a copy number ratio ranging between 0.01:1 and 1:1, 0.02:1 and 1:1, 0.05:1 and 1:1, 0.08:1 and 1:1, 0.1:1 and 1:1, 0.2:1 and 1:1, 0.3:1 and 1:1, 0.5:1 and 1:1, 0.7:1 and 1:1, and 0.9:1 and 1:1 of the first expression vector and the second expression vector.
  • a copy number ratio ranging between 0.01:1 and 1:1, 0.02:1 and 1:1, 0.05:1 and 1:1, 0.08:1 and 1:1, 0.1:1 and 1:1, 0.2:1 and 1:1, 0.3:1 and 1:1, 0.5:1 and 1:1, 0.7:1 and 1:1, and 0.9:1 and 1:1 of the first expression vector and the second expression vector.
  • an improved lentivirus is characterized by: reduced resistance onset, increased stability, reduced serum inactivation, increased titer, increased infectivity or any combination thereof, in a subject administered therewith, compared to a control lentivirus.
  • a method for improving in vivo gene therapy targeting a hematopoietic cell in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a lentivirus comprising: (a) a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) positions 47-50; (iii) positions 184-189; (iv) positions 349-353; and (v) any combination of (i) to (iv), compared to a wildtype VSV-G; and (b) a second nucleic acid sequence comprising a sequence encoding an antibody VH; a sequence encoding an antibody VL; and a sequence encoding a transmembrane domain, wherein the encoded VH and VL form a scFv, wherein the lentivirus is obtained
  • the method further comprises a step preceding the administering, comprising preparing the lentivirus as disclosed herein.
  • improving comprises: reducing resistance of the subject to the lentivirus, increasing abundance and/or stability of the lentivirus in the subject, reducing inactivation (e.g., proteolytic digestion) of the lentivirus in serum of the subject, or any combination thereof, compared to a control lentivirus.
  • inactivation e.g., proteolytic digestion
  • a control lentivirus is not the lentivirus of the invention.
  • a control lentivirus comprises a WT VSV-G.
  • a control lentivirus comprises a mutated VSV-G being devoid of the mutation(s) disclosed herein.
  • a control lentivirus is devoid of a scFv.
  • a control lentivirus is devoid of a scFv as disclosed herein.
  • a method for introducing a nucleic acid molecule of interest predominantly or specifically into a hematopoietic cell or a progenitor thereof in a subject in need thereof is provided.
  • the method comprises the steps of: (a) providing a blood sample obtained or derived from the subject; (b) contacting the blood sample with a vector as disclosed herein; (c) determining the rate of vector inactivation in the blood sample of step (b), wherein a rate of vector inactivation being below a predetermined threshold is indicative of the subject being suitable for in vivo hematopoietic gene therapy treatment, thereby for determining the suitability of a subject for in vivo hematopoietic gene therapy treatment.
  • a rate of vector inactivation being equal to or above a predetermined threshold is indicative of the subject being unsuitable for in vivo hematopoietic gene therapy treatment, as disclosed herein.
  • a method for treating a subject afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor cell thereof.
  • step (a) of the method disclosed herein comprises obtaining a blood sample from a subject.
  • the method comprises administering to the subject an effective amount of: (a) the expression vector disclosed herein; (b) the lentivirus disclosed herein; or (c) the composition disclosed herein; and a nucleic acid molecule of interest, thereby introducing a nucleic acid molecule of interest predominantly or specifically into a hematopoietic cell or a progenitor thereof of the subject.
  • the therapeutic nucleic acid molecule is configured to substituting or rectifying a genetic loss of function of a blood cell or a progenitor cell thereof, thereby treating the subject afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor cell thereof.
  • the therapeutic nucleic acid molecule is configured to introducing a therapeutic gene product to a blood cell or a progenitor cell thereof, thereby treating a subject afflicted with a genetic disease.
  • introducing is into the genome of a hematopoietic cell or a progenitor thereof, in the subject.
  • the hematopoietic cell or the progenitor cell is characterized by CD34 expression, CD90 expression, or both.
  • expression comprises presence or increased abundance of an mRNA transcript of the CD34 gene, the CD90 gene, or both, presence or increased abundance of a protein product translated therefrom, or any combination thereof.
  • a hematopoietic cell comprises a stem cell.
  • a stem cell is a hematopoietic stem cell.
  • a hematopoietic cell comprises a T cell or a NK cell.
  • stem cell is used herein to refer to a cell (e.g., a vertebrate stem cell) that has the ability both to self-renew and to generate a differentiated cell type (see Morrison et al. (1997) Cell 88:287-298).
  • Stem cells may be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers.
  • Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny.
  • hematopoietic stem cell or “HSC” refer to an undifferentiated hematopoietic cell that is capable of differentiating into all blood cell types, myeloid and lymphoid cells.
  • the HSC may reside in the bone marrow or be found elsewhere e.g., peripheral blood.
  • the hematopoietic cell or the progenitor cell comprises at least one genetic aberration conferring a disease.
  • the genetic aberration comprises a mutation, deletion, insertion, inversion, translocation, or any combination thereof.
  • the hematopoietic cell or the progenitor cell comprises at least one loss of function mutation.
  • the mutation is inducing, resulting, or conferring a disease.
  • the loss of function mutation comprises a nonsense mutation or missense mutation.
  • predominantly or specifically is at least by 2-fold, 3-fold, 5-fold, 10- fold, 20-fold, 30-fold, 50-fold, 70-fold, 100-fold, 250-fold, 350-fold, 500-fold, 750-fold, or 1,000- fold, compared to control, or any value and range therebetween.
  • Each possibility represents a separate embodiment of the invention.
  • a control comprises a non-hematopoietic cell or any progenitor thereof.
  • a control comprises a cell devoid of expression or characterized by under-expression or downregulation of CD34, CD90, or both.
  • a control comprises a cell having a cell surface devoid of CD34, CD90, or both.
  • administering comprises intravenously administering.
  • administering comprises locally administering.
  • administering comprises intravenously administering and locally administering.
  • administering comprises intraosseous infusion.
  • administering comprises intrathymically administering.
  • administering comprises intrathecally administering.
  • transducing and/or introducing comprises transferring an expression vector comprising the polynucleotide molecule into a target cell.
  • a target cell comprises or is a hematopoietic cell or a progenitor thereof.
  • transferring is or comprises transfecting. In some embodiments, transferring is or comprises lipofecting. In some embodiments, transferring is or comprises nucleofecting. In some embodiments, transferring is or comprises viral infection.
  • treatment or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.
  • administering refers to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.
  • the terms “subject” or “individual” or “animal” or “patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.
  • a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ⁇ 100 nm.
  • viruses containing wt or mut VSV-G were produced. Briefly, 293T cells were co-transfected with the lentiviral vector pCCL-PGK-GFP, plasmids encoding viral gag, pol, and rev genes and the wt or mutVSV-G. For transfection, the TransIT-VirusGEN® Transfection Reagent (Minis) was used. Generated LVs were collected and filtered 48 hours after transfection. LVs titers were determined using the Lenti- X p24 Rapid Titer Kit (Takara Bio cat. 632200).
  • VSV-G mutant titers were approximately 0.7-fold of wt VSV-G.
  • Transduction of 293T or U937 cells was carried out by mixing the viruses with polybrebe (Sigma) transduction enhancer.
  • polybrebe Sigma
  • transduction was performed in the presence of the LentiBOOST (sirion biotech) transduction enhancer. Twenty-four (24) hours later, cells were washed to remove any virions left in the media. Cells were maintained and collected for analysis 4-5 days post transduction. To evaluate co- exitance of binding and fusion events of the transfected cells, percentage of positive GFP cells were measured using flow cytometry. No reduction in infectivity was measured in mutants H49A and E353P.
  • K50T and T352A mutants and wt VSV-G plasmids were transfected with equal DNA amounts to 293T cells. Twenty-four (24) hours post transfection, cells were collected and stained with anti- VSV-G (Abeam ab3863) to measure VSV-G expression levels by flow cytometry (Fig. 5).
  • VSV-G plasmid concentration was 100 ng/pl and tested scFv concentrations were set to 200 ng/pl ,100 ng/pl, 50 ng/pl and 25 ng/pl corresponding to 1:2, 1:1, 2:1 and 4:1 VSV-G to scFv ratios.
  • the scFvs included in this set of experiments were anti-CD90 and anti-CD34 5B 12 and 2E10 clones, respectively.
  • the 1:1 and 2:1 VSV-G to scFv ratios demonstrated the best co-expression levels (Fig. 6). Therefore, the 1:1 ratio (100 ng/pl of each plasmid) was chosen for further investigation and LV production.
  • 293T stable cell lines were prepared. Briefly, 293T cells were transduced with CD90+_puromycin, CD34+_puromycin or MPL+_puromycin LVs in the presence of polybrene transfection enhancer. Twenty-four (24) hours later, cells were washed to remove any virion debris. Cells were maintained in puromycin free DMEM for 3 days to recover and then incubated in DMEM containing puromycin for selection. To confirm CD90+/CD34+/MPL+ expression, samples from the stable cell lines were collected and incubated with antibodies against CD90, CD34 and MPL respectively.
  • Flow cytometry was used to demonstrate successful markers expression (Fig. 7A-7C).
  • the generated cell lines were transduced with LV- scFv4C8-VSVG K50T - T352A , LV scFvCD90-VSVG K50T - T352A or LV- scFvMPL-VSVG K50T T352A .
  • the parental cell lines were used.
  • VSVG K50T - T352A as well as scFv LVs were used as controls (data not shown).
  • LV-VSVG WT was used as a reference for efficient transduction.
  • Transduction was carried out by mixing the viruses with polybrene (Sigma) transduction enhancers into an appropriate tissue culture dish. The cells, in which puromycin was removed for transduction, were added to the well at a concentration of 0.5- IxlO 6 cells/ml. Twenty-four (24) hours later, cells were washed to remove any virions left in the media. Cells were maintained and collected for analysis 5 days post transduction.
  • infectivity level of cells transduced with scFv4C8-VSVG K50T - T352A an d scFv5B12-VSVG K50T T352A LVs were 0.97 and 0.2-fold in comparison to cells transduced with VSVG wt LV (Fig. 8B).
  • 293T+CD34 cells were co-cultured and transfected with either VSVG wt or scFv4C8-VSVG K50T - T352A LVs.
  • scFv -VSVG K50T T352A LV To evaluate the ability of the scFv -VSVG K50T T352A LV to transduce primary human cells, mobilized peripheral blood cells (MPBCs) derived CD34+ human hematopoietic stem cells (HSCs) and peripheral blood mononuclear cells (PBMC) derived CD3+ human T cells were used.
  • G-CSF Granulocyte colony stimulating factor
  • CD34+ cells are purified by CD34 positive selection microbeads (Miltenyi). T cells were purified using the human Pan T cell isolation kit (Millteny). Cell purity was measured using flow cytometry (93% CD34+ and 98% CD3+ cells; data not shown).
  • T cells were activated using TransAct (Millteny) according to the manufacturer's instructions.
  • TransAct Millteny
  • resting and activated human T cells were cultured in LymphoONE T cells expansion media (Takara) supplemented with IL-2 (PeproTech) and human serum (sigma) for 48h prior to transduction
  • human G-CSF MPBC-derived CD34+ cells were cultured in serum- free SCGM medium (CellGenix) supplemented with a cocktail of cytokines: IL-3, TPO, SCF, and FLT-3L (PeproTech) for minimal incubation prior to transduction.
  • Transduction was carried out by mixing the viruses with the LentiBOOST (Sirion) transduction enhancer into an appropriate tissue culture dish.
  • CD3+ or CD34+ cells were added to the well at a final concentration of 0.9xl0 6 cells/ml. Twenty-four (24) hours later, cells were washed to remove any virions left in the media. Cells were maintained and collected for analysis 4 days post transduction. Transduced cells were evaluated by FACS with the following markers: CD34+, CD3+, and GFP. To demonstrate specificity in CD34+, cells were gated based on CD34 level (Fig. 9A). While no difference was observed in both population infectivity rates when VSVG WT LV was used, a clear and significant difference of 71-fold was measured in cells transduced with scFv- VSVG K50T - T352A LV (Fig 9B).
  • VSVG WT LV injection results in high transduction rate in hepatocytes, partly due to the high level of LDL-R on these cells (Milani et al., 2022; 2019).
  • HUH7 and HepG2 hepatocytes cell lines were transduced with either VSVG WT LV or 4C8 - VSVG K50T T352A LV in the presence of polybrene (Sigma) transduction enhancer.
  • VSV-G and Cocal resistance to complement-mediated inactivation was tested by preincubation of five-fold diluted VSV-G and Cocal pseudotyped LVs in fresh or heat inactivated (HI) human serum for 1 hour at 37 °C (QUIDEL, Al 13). Following virus-serum incubation, 293 cells were infected at multiplicity of infection 1 (MOI1) and viral infectivity was measured by flow cytometry 3 days post transduction. Infectivity levels post incubation in serum were dramatically reduced by 7.6- and 6.7-fold using VSV-G and Cocal LVs, respectively (Fig. 11A). As opposed to previous publications, we did not observe a substantial difference in serum sensitivity between of VSV-G and the Cocal pseudotyped LVs.
  • MOI1 multiplicity of infection 1
  • VSV-G expression plasmid 100, 50, and 25 ng/pl VSV-G, Fig. 11B
  • LV with decreasing VSV-G content were then incubated in human sera as described in the previous section. Titer recovery as measured by infectivity showed increased resistance to inactivation in human sera in correlation with reduced VSV-G levels (Fig. 11C).
  • the scFv-non-LDL-R binding LV maintains high infectivity levels at lower VSV-G concentrations in comparison to VSVG WT LV
  • VSV-G expression plasmid 100, 50, and 25 ng/pl
  • LVs were transduced into CD90 positive or negative 293T cells.
  • scFv _VSV-G WT LVs are used to transduce 293T CD90’ cells, binding solely depends on the LDL-R.
  • 293T CD90 + cells are transduced, binding can occur either from the LDL-R or the CD90 marker.
  • VSV-G T352A binding to CD90 + is mediated through the CD90 marker.
  • VSV-G is solely required for membrane fusion and not binding, potentially demanding lower levels of expression in comparison to when both binding and fusion are VSV-G dependent. Indeed, infectivity depended on VSV-G levels were better maintained in LVs expressing the VSV- G T352A , probably due to its ability to bind the cell through the scFv targeted against CD90 and not through the VSV-G mediated LDL-R binding (Fig. 12).
  • the K50T and T352A increase VSV-G serum resistance levels
  • VSV-G concentration reduced serum sensitivity was measured, while infectivity levels were still maintained (Fig. 12). Therefore, this concentration was determined as the optimal level for further investigation.
  • scFvCD90 _VSV-G T352A and VSV-G WT LVs were tested for human serum inactivation by incubating the virions with human serum (QUIDEL, Al 13) at 37 °C for 1 hr. Next, the incubated virions were transduced into 293T_ CD90 + cells and GFP levels were measured by flow cytometry to determine infection level.
  • the scFvCD90_VSV-G T352A was 2-fold more resistance to human serum in comparison to VSV-G WT LVs (Fig. 13).

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Abstract

The present application, in some embodiments, is directed to a synthetic polynucleotide including a nucleic acid sequence encoding a mutated vesicular stomatitis virus G (VSV-G) protein, being characterized by having reduced or no binding affinity to a low-density lipoprotein (LDL) receptor, compared to a control VSV-G. Further provided is a viral vector including the mutated VSV-G protein, and optionally further includes a single-chain variable fragment (scFv). Further provided are, inter alia, compositions including the expression vector, and use thereof, such as for in vivo gene therapy in a subject in need thereof.

Description

LENTIVIRAL VECTORS FOR IN VIVO TARGETING OF IMMUNE CELLS
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[001] The contents of the electronic sequence listing (NTX-P-002-PCT.xml; size: 59,327 bytes; and date of creation: March 05, 2023) is herein incorporated by reference in its entirety.
CROSS-REFERENCE TO RELATED APPLICATIONS
[002] This application claims the benefit of priority of Israel Patent Application No. 291148, titled "LENTIVIRAL VECTORS FOR IN VIVO TARGETING OF HEMATOPOIETIC CELLS", filed March 6, 2022, the contents of which are incorporated herein by reference in their entirety.
FIELD OF INVENTION
[003] The present invention is directed to lentiviral vectors optimized for in-vivo cell specific targeted transduction.
BACKGROUND
[004] Self-inactivating 3rd generation lentiviral vectors (LVs) stably integrate into the chromosomes of their targets, have a relatively large cloning capacity, and can transduce both dividing and nondividing cells. These synthetic viruses are now commonly used for ex vivo gene therapy (GT) applications, such as autologous hematopoietic stem cell (HSC) therapy, chimeric antigen receptor T cell therapy, and others. In this ex vivo process, HSCs or T cells are isolated from the patient, transduced by LVs, and then readministered to the patient.
[005] Ex vivo genetic modification is complex and requires sophisticated facilities and skilled personnel, which makes this a high-cost personalized treatment. Despite the drawbacks of the technology, ex vivo LV GT has proven to be safe and efficient with unparalleled ability to safely introduce large DNA segments to the genome of hematopoietic stem cells and T cells. Now, there is a pressing need to adapt this system for in-vivo gene delivery. In-vivo LV GT by direct injection of the gene-correcting vector into the blood stream of a subject holds a great promise for many reasons. In-vivo administration of the vector omits the labor-intensive and costly ex vivo process. Further, In-vivo LV GT would reduce the loss of “sternness” of HSCs, therefore, will increase homing/engraftment capacity as there is no need to culture the target cells. Lastly, in-vivo LV GT has the potential to eliminate the need for conditioning, thus, dramatically decreasing side effects of the procedure. Accordingly, in-vivo LV GT will be more readily distributed to the patients, and potentially will even allow therapy re-administration as required.
[006] Two main challenges for in vivo LV GT include: (1) lack of specific cell targeting; and (2) serum inactivation of the virus envelope glycoprotein mediated by the complement system.
[007] Cell targeting - There is a need to modify the target cell binding capacity of the LV system from its current ability of the vesicular stomatitis virus G (VSV-G) pseudotyped envelope protein to infect almost every cell type to targeting moieties that can target specific cell populations.
[008] Vesicular stomatitis virus (VSV) is a negative- strand RNA virus, and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. G plays a critical role during the initial steps of virus infection. First, it is responsible for viral attachment to specific receptors. After binding, virions enter the cell by a Clathrin-mediated endocytic pathway. In the acidic environment of the endocytic vesicle, G triggers the fusion between the viral and endosomal membranes, which releases the viral genome into the cytosol, initiating the subsequent steps of infection. Fusion is catalyzed by a low-pH-induced large structural transition from a pre-fusion to a post-fusion conformation, which are both trimeric.
[009] Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV-G. Nikolic et al have recently demonstrated that VSV-G is able to independently bind two distinct CR domains (CR2 and CR3) of LDL-R and report crystal structures of VSV-G in complex with those domains. In addition, they were able to identify specific segments which are important for binding. Specifically- residues 8 to 10 and 350 to 354 in the trimerization domain (TrD), 180 to 184 in the pleckstrin homology domain (PHD) and 47 to 50 in segment S2. (PCT EP2018/075824 and Nikolic et al; Nature Communications volume 9, Article number: 1029 (2018)).
[010] The use of LDL-R and its family members is not universal to all vesiculoviruses. While the close phylogenetic relatives to VSV such as Cocal, and Maraba viruses also interact with LDL-R, it has been shown that Piry and Chandipura viruses do not. Here we identified novel binding mutations of VSV-G, demonstrating reduced binding capabilities without effecting the membrane fusion capacity. To identify putative residues, we aligned and compared different vesiculoviruses family members both positive and negative LDL-R interactors. This comparison revealed conserved residues among the LDL-R binders family members which were not conserved among the family members utilizing other receptors for entry. To test for mutants binding capacity, 293T, U937, and human CD34+ and T cells transduction rates were compared to wt VSV-G. [Oi l] Ultimately to compensate for the loss of cell binding, scFvs aimed at surface cell markers expressed on different hematopoietic cells were introduced on the membrane of the viral vector. Transduction of target cells is achieved in two steps: (i) Target cell binding via selected scFvs; and (ii) fusion to the endosome membrane by the non-binding VSV-G mutans. This enables the targeting of specific hematological cell populations such as HSCs, T or NK cells in vivo.
[012] Serum inactivation - VSV-G serum inactivation was first described over 20 years ago. One option to address this challenge is by replacement of the attachment glycoprotein with that of other viruses. For example, international patent application No. PCT/US2010/033616 teaches Cocal virus pseudotyped LVs with increased serum resistance.
[013] Alternatively, several approaches have been explored to increase serum resistance of LVs. Inhibitors of complement improve vector survival in serum in-vitro. However, systemic delivery of complement inhibitors can be accompanied by toxicity. Incorporating complement regulatory proteins (CRPs) directly into a virus - including human decay-accelerating factor (DAF)/CD55 and or membrane cofactor protein (MCP)/CD46 - enhanced the resistance of the virus to human serum inactivation. However, the efficiency of CRP incorporation into virions varied with the types of virus and producer cell, and the direct fusion of CRP to viral proteins usually results in lowering viral titers. As another approach, chemical “shielding” of VSV-G via bioconjugation of polyethylene glycol (PEG) or Polyethylenimine enhanced the serum resistance of lentiviral vectors, though vector titer was reduced. Moreover, chemical modification adds an additional step in vector production for clinical development.
[014] There is still a great need for improved lentiviral vectors for in vivo targeting of immune cells.
SUMMARY
[015] The present invention, in some embodiments, discloses increasing serum resistance by optimizing the ratio of the envelope protein (e.g., VSV-G, cocal , RD114) and binding moieties (such as scFv against CD90 or CD34) on the viral membrane. Optimization of this ratio allows increasing serum resistance while maintaining high infectivity rates of a selected target cell population. Furthermore, the present invention, in some embodiments, is based, at least in part, on the surprising results showing that LV s expressing the novel reduced LDL receptor binding variants have increased resistance to human serum. [016] According to one aspect, there is provided a method for preparing an improved lentivirus targeting a hematopoietic cell for in vivo gene therapy, the method comprising: contacting a cell with: (a) a first expression vector comprising a nucleic acid sequence encoding a mutated vesicular stomatitis virus G protein (VSV-G), the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G; and (b) a second expression vector comprising a nucleic acid sequence comprising: (i) a nucleic acid sequence encoding an antibody heavy chain variable region (VH); (ii) a nucleic acid sequence encoding an antibody light chain variable region (VL); and (iii) a nucleic acid sequence encoding a transmembrane domain, wherein the encoded VH and VL form a single-chain variable fragment (scFv), wherein the contacting is at a copy number ratio ranging between 0.01:1 and 1:1 of the first expression vector and the second expression vector; and culturing the cell under conditions such that the nucleic acid sequence of the first expression vector and the nucleic acid sequence of the second expression vector are expressed, thereby, preparing the improved lentivirus targeting a hematopoietic cell for in vivo gene therapy.
[017] According to another aspect, there is provided a method for improving in vivo gene therapy targeting a hematopoietic cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lentivirus comprising: (a) a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G; and (b) a second nucleic acid sequence comprising a sequence encoding an antibody VH; a sequence encoding an antibody VL; and a sequence encoding a transmembrane domain, wherein the encoded VH and VL form a scFv, wherein the lentivirus is obtained or derived from a cell comprising the first nucleic acid sequence and the second nucleic acid sequence at a copy number ratio of ranging between 0.01:1 and 1:1.
[018] According to another aspect, there is provided a synthetic polynucleotide comprising a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G.
[019] According to another aspect, there is provided an expression vector comprising the synthetic polynucleotide disclosed herein. [020] According to another aspect, there is provided a viral vector comprising the expression vector disclosed herein.
[021] According to another aspect, there is provided a cell comprising: (a) the synthetic polynucleotide disclosed herein; (b) the expression vector disclosed herein; (c) the viral vector disclosed herein; (d) the lentivirus disclosed herein; or (e) any combination of (a) to (d).
[022] According to another aspect, there is provided a composition comprising: (a) the synthetic polynucleotide disclosed herein; (b) the expression vector disclosed herein; (c) the viral vector disclosed herein; (d) the lentivirus disclosed herein; (e) the cell disclosed herein; or (f) any combination of (a) to (e); and an acceptable carrier.
[023] In some embodiments, the improved lentivirus is characterized by: reduced resistance onset, increased stability, reduced serum inactivation, increased titer, increased infectivity or any combination thereof, in a subject administered therewith, compared to a control lentivirus.
[024] In some embodiments, the improving comprises: reducing resistance of the subject to the lentivirus, increasing stability of the lentivirus in the subject, reducing inactivation of the lentivirus in serum of the subject, or any combination thereof, compared to a control lentivirus.
[025] In some embodiments, the administering is via a route selected from the group consisting of: intravenous, local, intraosseous infusion, intrathymical, intrathecal, and any combination thereof.
[026] In some embodiments, the mutated VSV-G further comprises at least one amino acid substitution of positions 47-50, compared to the wildtype VSV-G.
[027] In some embodiments, the mutated V SV -G comprises: (i) a substitution of lysine at position 50 (K50) to threonine; (ii) a substitution of threonine at position 352 (T352) to alanine; or (iii) both (i) and (ii).
[028] In some embodiments, the mutated VSV-G is characterized by having reduced or no binding affinity to a low-density lipoprotein (LDL) receptor, compared to a control VSV-G.
[029] In some embodiments, the scFv is characterized by having specific binding affinity to any one of cluster of differentiation CD 34, CD90, and both.
[030] In some embodiments, the nucleic acid sequence encoding the antibody VH comprises the nucleic acid sequence set forth in SEQ ID Nos: 3, 33, 37, or 11. [031] In some embodiments, the nucleic acid sequence encoding the antibody VL comprises the nucleic acid sequence set forth in SEQ ID Nos: 5, 34, 38, or 13.
[032] In some embodiments, the lentivirus comprises the nucleic acid sequence set forth in SEQ ID Nos: 6, 29, 3 l or 14.
[033] In some embodiments, the encoded VH comprises the amino acid sequence set forth in SEQ ID Nos: 7, 35, 39, or 9.
[034] In some embodiments, the encoded VL comprises the amino acid sequence set forth in SEQ ID Nos: 15, 36, 40 orl7.
[035] In some embodiments, the formed scFv comprises the amino acid sequence set forth in SEQ ID Nos: 10, 30, 32 or 18.
[036] In some embodiments, the encoded transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 19-22.
[037] In some embodiments, the subject is afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor thereof.
[038] In some embodiments, the synthetic polynucleotide further comprising a second nucleic acid sequence comprising: (a) a nucleic acid sequence encoding an antibody VH; (b) a nucleic acid sequence encoding an antibody VL; and (c) a nucleic acid sequence encoding a transmembrane domain, wherein the encoded VH and VL form a scFv.
[039] In some embodiments, the expression vector is a lentivirus-based expression vector.
[040] In some embodiments, the viral vector is a lentivirus.
[041] In some embodiments, the scFv is integrated into a viral envelope of the lentivirus via the transmembrane domain.
[042] In some embodiments, the carrier is a pharmaceutically acceptable carrier.
[043] In some embodiments, the composition is for use in in vivo gene therapy in a subject in need thereof.
[044] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
[045] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[046] Fig. 1 includes a non-limiting schematic description of a single-chain variable fragment (scFv) construct as disclosed herein. Signal peptide, SP; Variable heavy, VH; Linker; Variable light, VL; Transmembrane domain, TM; Cytoplasmatic tail, CT; and Linker, L.
[047] Figs. 2A-2B include vertical bar graphs. (2A) A vertical bar graph showing CD90 scFv expression on 293T cell membrane. Each of the 8 different plasmid constructs containing the CD90 scFv fused to a different signal peptide and a transmembrane domain were co transfected with a GFP plasmid into 293T cells. Twenty-four hours post transfection cells were harvested and the scFv expression on the cell membrane was measured by flow cytometry using an anti-Myc antibody. Transfection efficiency was even in all tested constructs as determined by GFP expression (data not shown). The highest Myc expression was observed in constructs #7 and #8 harboring the CD8 transmembrane domain and the signal peptide of the murine IgK chain and VSV- G respectively. (2B) A vertical bar graph showing plasmid constructs containing the different scFV clones expression on 293T cell membrane. All constructs harboring the CD8 transmembrane domain and the signal peptide of the VSVG were co-transfected with a GFP plasmid into 293T cells. Twenty-four hours post transfection cells were harvested and the scFv expression on the cell membrane was measured by flow cytometry using an anti-Myc antibody. Myc expression was observed in all constructs ranging between 39.5 - 82% of positive cells.
[048] Fig. 3 includes a multiple sequence alignment of VSV (VSIVA), Cocal (COCAV), Maraba (MARAV), Piry (PIRYV) and Chandipura (CHAV) glycoproteins. The consensus sequence is highlighted in black bold. Matched amino acid are marked by a gray background. CR2-CR3 binding segments marked with black rectangles. [049] Figs. 4A-4C include vertical bar graphs showing defected mutants infection capacity in comparison to wt VSV-G in different cells. Fold change infectivity in (4A) 293T, (4B) U937, and (4C) human primary CD34+ and activated T cells post transduction with WT or mutants VSV-G.
[050] Fig. 5 includes plots of flow cytometry (FACS) analysis showing VSV-G mutants (K50T, and T352A) expression on 293T cells membrane in comparison to wt VSV-G. Each of the plasmid constructs bearing a mutation in the VSVG protein was transfected into 293T cells. Twenty-four hours post transfection cells were harvested and VSV-G expression on the cell membrane was measured by flow cytometry using an anti-VSV-G antibody.
[051] Fig. 6 includes a vertical bar graph showing percentage of double positive VSV-G and CD90 scFv expression. 293T cells were transfected with VSV-G (100 ng/pl) and CD90 scFv (100, 50 and 25 ng/pl). Twenty-four hours post transfection cells were harvested and VSV-G and CD90 scFv expression on cell membrane was measured by flow cytometry using anti-VSV-G and anti- Myc antibodies, respectively.
[052] Figs. 7A-7C includes flow cytometry plots characterizing (7A) 293T_CD90+; (7B) 293T_CD34+; and (7C) 293T_MPL+ stable cell lines. After puromycin selection, the stable cell lines were incubated with antibodies against CD90, CD34 and MPL respectively. Flow cytometry was used to demonstrate successful markers expression.
[053] Figs. 8A-8E includes vertical bar graphs and flow cytometry plots showing infection capacity of scFv with VSV-G K50T T352A in comparison to VSV-GWT in 293T ± marker and Jurkat cells. Cells were transduced with the different LVs and 3 days post transduction, and infection rates were measured. (8A) A vertical bar graph showing scFv 90 _VSV-G K50T T352A in comparison to VSV-GWT in 293T ± CD90. (8B) A vertical bar graph showing scFv 34 _VSV-G K50T-T352A in comparison to VSV-GWT in 293T ± CD34. (8C) Flow cytometry plots showing scFv4C8_VSV-G K5OT_T352A jn comparison (o VSV-GWT in co-culture of 293T ± CD34. (8D) A vertical bar graph showing scFv CD3 _VSV-G K50T T352A in comparison to VSV-GWT in Jurkat cells. (8E) A vertical bar graph showing scFv 1.61 _VSV-G K50T T352A in comparison to VSV-GWT in 293T ± MPL.
[054] Figs. 9A-9C includes vertical bar graphs and flow cytometry plots showing infection capacity of VSV-G WT, scFv4C8_VSV-G K50T-T352A and scFvCD3 VSV-G K50T-T352A in primary HSC gated based on CD34 levels (high and low), activating and resting T cells. Cells were transduced with the different LVs and 4 days post transduction; infection rates were measured. (9A) A flow cytometry plot sowing CD34 cells gating strategy. (9B) A vertical bar graph showing scFv 4C8 _VSV-G K50T-T352A in comparison to VSV-GWT in CD34High and CD34Low cells. (9C) Flow cytometry plots showing VSV-G K50T-T352A and scFvCD3_VSV-G K50T-T352A in comparison to VSV-GWT in activating and resting T cells.
[055] Figs. 10A-10B include vertical bar graph showing infection capacity of VSV-G WT and scFv4C8_VSV-G K50T-T352A in (10A) hepatocytes cell lines and (10B) florescent microscopy images in mouse primary hepatocytes.
[056] Figs. 11A-11C include vertical bar graphs and flow cytometry plots. (11A) Infectivity levels post incubation in serum was dramatically reduced to 7.6- and 6.7-fold reduction using VSV- G and cocal LVs, respectively. (11B) VSV-G membrane expression is reduced with lower VSV-G plasmid amount transfection. (11C) Increased resistance to inactivation in human sera in correlation with reduced VSV-G plasmid levels (100, 50, and 25 ng/pl VSV-G plasmid). Heated inactivated (HI).
[057] Fig. 12 includes a graph showing that the infectivity level of WT VSV-G is more sensitive to reduction in VSV-G levels compared to T352A mut VSV-G.
[058] Fig. 13 includes a vertical bar graph showing that the VSV-GWT is more sensitive to human serum compared to scFvVSV-GT352A.
DETAILED DESCRIPTION
[059] According to one aspect, there is provided a method for preparing an improved lentivirus targeting a hematopoietic cell for in vivo gene therapy.
[060] In some embodiments, the method comprises contacting a cell with: (a) a first expression vector comprising a nucleic acid sequence encoding a mutated vesicular stomatitis virus G protein (VSV-G), the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) positions 47-50; (iii) positions 184-189; (iv) positions 349-353; and (v) any combination of (i) to (iv), compared to a wildtype VSV-G; and (b) a second expression vector comprising a nucleic acid sequence comprising: (i) a nucleic acid sequence encoding an antibody heavy chain variable region (VH); (ii) a nucleic acid sequence encoding an antibody light chain variable region (VL); and (iii) a nucleic acid sequence encoding a transmembrane domain, wherein the encoded VH and VL form a single-chain variable fragment (scFv), wherein the contacting is at a copy number ratio ranging between 0.01:1 and 1:1 of the first expression vector and the second expression vector; and culturing the cell under conditions such that the nucleic acid sequence of the first expression vector and the nucleic acid sequence of the second expression vector are expressed. [061] In some embodiments, the improved lentivirus is characterized by: reduced resistance onset, increased stability, reduced serum inactivation, improved titer, improved infectivity, or any combination thereof, in a subject administered therewith, compared to a control lentivirus.
[062] According to another aspect, there is provided a method for improving in vivo gene therapy targeting a hematopoietic cell in a subject in need thereof.
[063] In some embodiments, the method comprises administering to a subject a therapeutically effective amount of a lentivirus comprising: (a) a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) positions 47-50; (iii) positions 184-189; (iv) positions 349-353; and (v) any combination of (i) to (iv), compared to a wildtype VSV-G; and (b) a second nucleic acid sequence comprising a sequence encoding an antibody VH; a sequence encoding an antibody VL; and a sequence encoding a transmembrane domain, wherein the encoded VH and VL form a scFv, wherein the lentivirus is obtained or derived from a cell comprising the first nucleic acid sequence and the second nucleic acid sequence at a copy number ratio of ranging between 0.01:1 and 1:1.
[064] In some embodiments, improving comprises: reducing resistance of the subject to the lentivirus, increasing stability and/or abundance of the lentivirus in the subject, reducing inactivation (such as by proteolytic enzyme(s) and/or protein(s), such as complement system proteins, but not limited thereto) of the lentivirus in serum of the subject, or any combination thereof, compared to a control lentivirus.
[065] In some embodiments, the contacting is at a copy number ratio ranging between: 0.01 : 1 and 1:1, 0.1:1 and 1:1, 0.05:1 and 1:1, 0.2:1 and 1:1, 0.3:1 and 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.01:0.99 and 0.1:0.9 of the first expression vector and the second expression vector. Each possibility represents a separate embodiment of the invention.
[066] In some embodiments, the first and second nucleic acid sequences as disclosed herein, are located on a single expression vector. In some embodiments, the first and second nucleic acid sequences as disclosed herein, are located on separate expression vectors. In some embodiments, the first and second nucleic acid sequences as disclosed herein, are located on separate expression vectors, being the first and second expression vectors disclosed herein.
[067] According to some embodiments, there is provided a synthetic polynucleotide comprising a nucleic acid sequence encoding a mutated vesicular stomatitis virus G (VSV-G) protein. In some embodiments, the mutated VSV-G protein is characterized by having reduced or no binding affinity to a low-density lipoprotein (LDL) receptor. [068] According to another aspect, there is provided a synthetic polynucleotide comprising a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) positions 47-50; (iii) positions 184- 189; (iv) positions 349-353; and (v) any combination of (i) to (iv), compared to a wildtype VSV- G.
[069] In some embodiments, the mutated VSV-G further comprises at least one amino acid substitution of positions 47-50, compared to a wildtype VSV-G.
[070] In some embodiments, the synthetic polynucleotide further comprises a second nucleic acid sequence comprising: a nucleic acid sequence encoding an antibody heavy chain variable region (VH); a nucleic acid sequence encoding an antibody light chain variable region (VL); and a nucleic acid sequence encoding a transmembrane domain.
[071] In some embodiments, the scFv is characterized by having specific binding affinity to any one of cluster of differentiation CD34, CD90, or both. In some embodiments, the scFv binds specifically to CD34, CD90, or both. In some embodiments, the scFv binds to CD34, CD90, or both, of a hematopoietic cell. In some embodiments, the scFv binds to CD34, CD90, or both, on the surface of a hematopoietic cell.
[072] In some embodiments, the antibody VH is encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID Nos: 3, 33, 37, or 11.
[073] In some embodiments, the antibody VL is encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID Nos: 5, 34, 38, or 13.
[074] In some embodiments, the synthetic polynucleotide further comprises a nucleic acid sequence encoding a linker being located between the nucleic acid sequence encoding the antibody VH and the nucleic acid sequence encoding the antibody VL.
[075] In some embodiments, the linker is encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 12 .
[076] In some embodiments, the synthetic polynucleotide comprises the nucleic acid sequence set forth in SEQ ID Nos: 6, 29, 31 or 14.
[077] In some embodiments, the encoded VH comprises the amino acid sequence set forth in SEQ ID Nos: 7, 35, 39, or 9.
[078] In some embodiments, the encoded VL comprises the amino acid sequence set forth in SEQ ID Nos: 15, 36, 40 orl7. [079] In some embodiments, the encoded linker comprises the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 16.
[080] In some embodiments, the formed scFv comprises the amino acid sequence set forth in SEQ ID Nos: 10, 30, 32 or 18.
[081] In some embodiments, the encoded transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 19-22.
[082] In some embodiments, the synthetic polynucleotide further comprises a nucleic acid sequence encoding a signal or a leading peptide.
[083] In some embodiments, the encoded signal or leading peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
[084] In some embodiments, the synthetic polynucleotide further comprises a promoter sequence, wherein any of the nucleic acid sequence encoding the antibody VH; the nucleic acid sequence encoding the antibody VL; and the nucleic acid sequence encoding the transmembrane domain is operably linked to the promoter sequence.
[085] In some embodiments, the synthetic polynucleotide further comprises at least one additional nucleic acid sequence encoding: a protein translation initiation motif, a tag, a cytoplasmic tail, a transcription termination motif, or any combination thereof.
[086] In some embodiments, the mutated VSV-G comprises a signal peptide. In some embodiments, the mutated VSV-G is devoid of a signal peptide.
[087] In some embodiments, when being devoid of a signal peptide, the mutated VSV-G comprises at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G.
[088] In some embodiments, when being devoid of a signal peptide, the mutated VSV-G further comprises at least one amino acid substitution in positions 47-50, compared to a wildtype VSV-G.
[089] In some embodiments, when comprising a signal peptide, the mutated VSV-G comprises at least one amino acid substitution in any one of: (i) positions 24-26; (ii) 200-205; (iii) 365-369; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G.
[090] In some embodiments, when comprising a signal peptide, the mutated VSV-G further comprises at least one amino acid substitution in positions 63-66, compared to a wildtype VSV-G. [091] In some embodiments, when being devoid of a signal peptide, the mutated VSV-G comprises a substitution of glutamic acid at position 352 (T352).
[092] In some embodiments, when being devoid of a signal peptide, the mutated VSV-G comprises a substitution of glutamic acid at position 352 (T352); and optionally a substitution of lysine at position 50 (K50), compared to a wildtype VSV-G.
[093] In some embodiments, when being devoid of a signal peptide, the mutated VSV-G comprises: (i) a substitution of glutamic acid at position 352 (T352) and (ii) a substitution of lysine at position 50 (K50), compared to a wildtype VSV-G.
[094] In some embodiments, when being devoid of a signal peptide, the mutated VSV-G comprises: (i) a substitution of lysine at position 50 (K50) to threonine (K50T); (ii) a substitution of threonine at position 352 (T352) to alanine (T352A); or (iii) both (i) and (ii), compared to a wildtype VSV-G.
[095] In some embodiments, when comprising a signal peptide, the mutated VSV-G comprises a substitution of threonine at position 368 (T368).
[096] In some embodiments, when comprising a signal peptide, the mutated VSV-G comprises a substitution of threonine at position 368 (T368), and optionally a substitution of lysine at position 66 (K66), compared to a wildtype VSV-G.
[097] In some embodiments, when comprising a signal peptide, the mutated VSV-G comprises: (i) a substitution of threonine at position 368 (T368); and (ii) a substitution of lysine at position 66 (K66), compared to a wildtype VSV-G.
[098] In some embodiments, when comprising a signal peptide, the mutated VSV-G comprises: (i) a substitution of lysine at position 66 (K66) to threonine (K66T); (ii) a substitution of threonine at position 368 (T368) to alanine (T368A); or (iii) both (i) and (ii), compared to a wildtype VSV- G.
[099] In some embodiments, wildtype VSV-G protein comprises the amino acid sequence: MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQV KMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQG TWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTV HNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACK MQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERIL DYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSR MVGMISGTTTEREEWDDWAPYEDVEIGPNGVERTSSGYKFPEYMIGHGMEDSDEHESSK AQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFL VERVGIHECIKEKHTKKRQIYTDIEMNREGK (SEQ ID NO: 41), or a functional analog thereof having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% thereto, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.
[0100] In some embodiments, wildtype VSV-G protein is encoded by the nucleic acid sequence: ATGAAGTGTCTGCTGTACCTGGCGTTCCTGTTTATCGGGGTGAACTGCAAGTTCACTA TCGTGTTTCCGCACAACCAAAAGGGCAACTGGAAAAACGTGCCTTCAAATTACCATT ATTGCCCCAGCAGCTCGGACCTGAACTGGCACAATGACCTCATTGGAACCGCGCTGC AGGTGAAGATGCCAAAGAGCCACAAGGCTATCCAGGCTGACGGATGGATGTGCCAC GCGTCAAAATGGGTGACTACCTGCGATTTCCGCTGGTACGGACCAAAATACATCACG CACAGCATCAGATCATTCACCCCGTCAGTGGAACAATGCAAAGAATCCATCGAACAG ACTAAGCAGGGAACCTGGCTGAACCCTGGATTTCCGCCGCAGTCGTGTGGGTACGCA ACCGTGACCGATGCAGAGGCCGTGATCGTGCAAGTCACGCCGCATCACGTGCTTGTG GACGAGTACACCGGAGAATGGGTCGATTCCCAGTTCATCAACGGCAAGTGCTCCAAC TACATTTGCCCAACCGTGCACAACAGCACTACTTGGCACAGCGACTACAAAGTGAAG GGTCTGTGTGATTCCAACCTGATCTCCATGGATATCACTTTCTTCTCGGAAGACGGCG AACTGTCCTCACTGGGCAAAGAAGGAACTGGGTTTCGCTCAAATTACTTCGCCTACG AAACTGGAGGAAAAGCCTGCAAGATGCAGTACTGCAAGCACTGGGGCGTGAGACTA CCCAGCGGTGTCTGGTTCGAGATGGCCGATAAGGACCTGTTTGCAGCAGCGAGATTC CCGGAATGCCCTGAGGGATCGAGCATCTCCGCTCCAAGCCAAACTTCAGTGGACGTG AGCCTGATCCAGGACGTGGAACGGATTCTCGACTACTCGCTGTGCCAGGAGACCTGG TCGAAGATCAGAGCGGGACTGCCCATCTCACCGGTGGACCTGTCCTACCTGGCGCCA AAGAATCCGGGCACTGGACCGGCGTTCACCATCATCAACGGCACCCTCAAATACTTC GAGACGCGGTACATCCGGGTGGACATCGCAGCTCCGATCCTCTCCCGGATGGTGGGA ATGATCTCGGGGACTACTACCGAACGCGAGCTCTGGGACGACTGGGCACCTTACGAG GATGTCGAGATCGGACCTAACGGAGTGCTCCGGACCTCCTCCGGGTACAAGTTCCCT CTGTACATGATCGGCCATGGCATGCTGGACTCGGATCTGCATCTGTCGTCCAAAGCA CAGGTGTTTGAACACCCACACATTCAAGACGCCGCCAGCCAGCTGCCGGACGATGAG TCGCTGTTCTTCGGAGACACGGGCTTGTCAAAGAATCCCATCGAGCTGGTGGAAGGA TGGTTTTCATCCTGGAAAAGCAGCATCGCTTCATTCTTCTTCATCATTGGCCTGATCA TCGGCCTATTTCTAGTCCTGCGGGTGGGAATTCATCTGTGCATCAAGCTCAAGCACAC TAAGAAGCGGCAAATCTACACTGATATCGAGATGAATCGCCTGGGCAAGTAG (SEQ ID NO: 43), or an analog thereof having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.
[0101] As used herein, the term "functional analog" encompasses any analog of VSV-G protein having the same or equivalent activity attributed to a VSV-G protein and having an amino acid sequence differing from the wildtype VSV-G protein by at least one amino acid.
[0102] According to some embodiments, the synthetic polynucleotide further comprises a second nucleic acid comprising: (a) a first nucleic acid sequence encoding an antibody heavy chain variable region (VH); (b) a second nucleic acid sequence encoding an antibody light chain variable region (VL); and (c) a third nucleic acid sequence encoding a transmembrane domain.
[0103] In some embodiments, the encoded VH and VL form a single-chain variable fragment (scFv).
[0104] According to some embodiments, there is provided a polynucleotide comprising: (a) a first nucleic acid sequence encoding an antibody heavy chain variable region (VH); (b) a second nucleic acid sequence encoding an antibody light chain variable region (VL); and (c) a third nucleic acid sequence encoding a transmembrane domain.
[0105] In some embodiments, the encoded VH and VL form a single-chain variable fragment (scFv) having specific binding affinity to cluster of differentiation CD34 or CD90.
[0106] In some embodiments, the polynucleotide disclosed herein, is an isolated polynucleotide. In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the DNA molecule is an isolated DNA molecule. In some embodiments, the polynucleotide or the DNA molecule is a complementary DNA (cDNA) molecule.
[0107] In some embodiments, the first nucleic acid sequence comprises the nucleic acid sequence: CAAGTGAAGCTGCAAGAGTCTGGCCCTGGACTGGTGCAGCCTAGCCAGAGCCTGAG CTTCATCTGTACCGTGTCCGGCTTCAGCCTGACATCTCATGGCGTGCACTGGGTCCGA CAGAGCCCTGGAAAAGGACTGCAGTGGCTGGGAGTGATTTGGGGAGCCGGCAGAAC CGATTACAACGCCGCCTTCATCAGCAGACTGAGCATCAGCCGGGACATCAGCAAGA GCCAGGTGTTCTTCAAGATGAACAGCCTGCAGGTCGACGACACCGCCATCTACTACT GCGCCCGGAACAGATACGAGAGCTACTTCGACTATTGGGGCCAGGGCACCACCGTG ACAGTTTCTAGC (SEQ ID NO: 3). [0108] In some embodiments, the first nucleic acid sequence comprises the nucleic acid sequence:
ATGGCTAGCGCTTCTCAGGTGCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCT
GGCGCCTCTGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCGGCTACTAC
GTGCACTGGGTCCGACAGGCTCCAGGACAAGGACTGGAATGGATGGGCTGGGTCAA
CCCCAATAGCGGCGACACCAATTACGCCCAGAAATTCCAGGGCAGAGTGACCATGA
CCAGAGACACCAGCATCAGCACCGCCTACATGGAACTGAGCGGCCTGAGATCCGAT
GACACCGCCGTGTACTACTGCGCCAGAGATGGCGACGAGGACTGGTACTTCGATCTG TGGGGCAGAGGCACCCCTGTGACAGTGTCTAGCGGAATCCTT (SEQ ID NO: 11).
[0109] In some embodiments, the first nucleic acid sequence comprises the nucleic acid sequence:
GAGATTCAGCTGCAGCAGTCTGGCCCCGAGCTTATGAAGCCTGGCGCCTCTGTGAAG
ATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCAGCTACTACATGCACTGGGTCAAG
CAGAGCCAGGGCAAGAGCCTGGAATGGATCGGCTTCATCGACCCCTTCAACGGCGG
CATCACCTACAACCAGAAGTTCAAGGGCAAAGCCACACTGACCGTGGACAGAAGCA
GCAGCACCGCCTATATGCACCTGAGAAGCCTGACCAGCGAGGACAGCGCCGTGTACT
ACTGCGCCAGATGCTACTACAACTACGACGACGAGGGCAGAGCCATGGACTATTGG GGCCAGGGAACAAGCGTGACCGTGTCTAGT(SEQ ID NO: 33).
[0110] In some embodiments, the first nucleic acid sequence comprises the nucleic acid sequence: ATGGCTGTTCTGGGCCTGCTGCTGTGCCTGGTCACCTTTCCAAGCTGTGTGCTGAGCC
AGGTGCAGCTGAAAGAGTCTGGACCTGGACTGGTGGCCCCTAGCCAGAGCCTGTCTA
TCACCTGTACCGTGTCCGGCTTCAGCCTGACCGATTACGGCGTGACCTGGATCAGAC
AGCCTCCTGGCAAAGGCCTGGAATGGCTGGGAGTTATTTGGGGCGGAGGCAGCACCT
ACTACAACAGCGCCCTGAAGTCCCGGCTGAGCATCAGCAAGGACAACAGCAAGTCC
CAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATGTACTACTGT
GCCAAGCACGAGAGCTACTACCCCTTCGTGTATTGGGGCCAGGGCACCCTGGTTACA GTGTCTGCT (SEQ ID NO: 37).
[0111] In some embodiments, the first nucleic acid sequence comprises the nucleic acid sequence set forth in SEQ ID NOs: 3, 11, 33, or 37.
[0112] In some embodiments, the second nucleic acid sequence comprises the nucleic acid sequence:
GATGTGCTGATGACCAACACACCCCTGAGCCTGCCTGTGTCTCTGGGAGATCAGGCC
AGCATCAGCTGCAGATCCAGCCAGAATCTGGTGCACAGCAACGGCAACACCTACCTG
CACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTAACCTGCTGATCTACAAGGTGTCC AACCGGTTCAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGAGTTC
ACCCTGAAGATCTCTAGAGTGCAGGCCGAGGACCTGGGCGTGTACTTCTGTAGCCAG
TCTACCCACGTGCCACTGACCTTTGGCGCCGGATCTAAGCTGGAACTGAAG (SEQ ID NO: 5).
[0113] In some embodiments, the second nucleic acid sequence comprises the nucleic acid sequence:
GACATTAGACTGACACAGAGCCCTAGCAGCCTGAGCGCCAGCATCGGAGACAGAGT
GACAATCACCTGTAGAGCCAGCCAGGGCATCAGCAGATCCCTCGTGTGGTATCAGCA
GAAGCCTGGCAAGGCCCCTCGGCTGCTGATCTATGCTGCTAGCACACTGCAGAGCGG
CGTGCCCTCTAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATATCT
AGCCTGCAGCCAGAGGACTTCGCCACCTACTACTGCCTGCAGCACAACACATACCCC
TTCACCTTCGGACCCGGCACCAAGGTGGACATCAAGTCTGGAATCCCCGAGCAGAAG CTG (SEQ ID NO: 13).
[0114] In some embodiments, the second nucleic acid sequence comprises the nucleic acid sequence:
GACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTCTGGGAGAAAGAGTG
TCCCTGACCTGTAGAGCCAGCCAGGATATCGGCAGCTCCCTGAATTGGCTGCAGCAG
GGACCTGACGGCACCTTCAAGAGACTGATCTACGCCACCTCCAGCCTGGATAGCAGC
GTGCCCAAGAGATTCAGCGGCAGCAGAAGCGGCAGCGACTACAGCCTGACAATCAG
CTCTCTGGAAAGCGAGGACTTCGTGGACTACTACTGCCTGCAGTACGCCAGCTCTCC CTACACATTTGGCGGAGGCACCAAGCTGGAAATCAAGCGG (SEQ ID NO: 34).
[0115] In some embodiments, the second nucleic acid sequence comprises the nucleic acid sequence:
GACGTGGTCATGACACAGACACCTCTGAGCCTGCCTGTGTCTCTGGGAGATCAGGCC
AGCATCAGCTGCAGAAGCAGCCAGAGCCTGATCAACAGCAACGGCAACACCTACCT
GCACTGGTATCTGCAGAAACCCGGACAGAGCCCCAAGCTGCTGATCCACAGAGTGTC
CAACAGATTCAGCGGCGTGCCCGACAGATTTTCTGGCAGCGGCTCTGGCACCGACTT
CACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACCTGGGCGTGTACTTCTGTAGCCA
GAGCACACACGTGCCCTGGACATTTGGCGGCGGAACAAAGCTGGAAATCAAGCGG (SEQ ID NO: 38).
[0116] In some embodiments, the second nucleic acid sequence comprises the nucleic acid sequence set forth in SEQ ID NOs: 5, 13, 34 or 38. [0117] In some embodiments, any one of the first and second polynucleotides, further comprises a nucleic acid sequence encoding a linker. In some embodiments, the nucleic acid sequence encoding a linker is located between the first nucleic acid sequence, and the second nucleic acid sequence of the second polynucleotide, of the polypeptide disclosed herein. In some embodiments, the linker is a peptide or a proteinaceous linker. In some embodiments, the linker is a flexible linker.
[0118] In some embodiments, the sequence nucleic acid sequence encoding a linker comprises the nucleic acid sequence:
GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 4).
[0119] In some embodiments, the nucleic acid sequence encoding a linker comprises the nucleic acid sequence:
GGATCTGGCGGCGGAGGAAGCGGAGGCGGAGGTTCTGGTGGTGGCGGCTCT (SEQ ID NO: 12).
[0120] In some embodiments, the nucleic acid sequence encoding a linker comprises the nucleic acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 12.
[0121] In some embodiments, the second polynucleotide comprises the nucleic acid sequence: CAAGTGAAGCTGCAAGAGTCTGGCCCTGGACTGGTGCAGCCTAGCCAGAGCCTGAG CTTCATCTGTACCGTGTCCGGCTTCAGCCTGACATCTCATGGCGTGCACTGGGTCCGA CAGAGCCCTGGAAAAGGACTGCAGTGGCTGGGAGTGATTTGGGGAGCCGGCAGAAC CGATTACAACGCCGCCTTCATCAGCAGACTGAGCATCAGCCGGGACATCAGCAAGA GCCAGGTGTTCTTCAAGATGAACAGCCTGCAGGTCGACGACACCGCCATCTACTACT GCGCCCGGAACAGATACGAGAGCTACTTCGACTATTGGGGCCAGGGCACCACCGTG ACAGTTTCTAGCGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTC TGATGTGCTGATGACCAACACACCCCTGAGCCTGCCTGTGTCTCTGGGAGATCAGGC CAGCATCAGCTGCAGATCCAGCCAGAATCTGGTGCACAGCAACGGCAACACCTACCT GCACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTAACCTGCTGATCTACAAGGTGTC CAACCGGTTCAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGAGTT CACCCTGAAGATCTCTAGAGTGCAGGCCGAGGACCTGGGCGTGTACTTCTGTAGCCA GTCTACCCACGTGCCACTGACCTTTGGCGCCGGATCTAAGCTGGAACTGAAG (SEQ ID NO: 6).
[0122] In some embodiments, the second polynucleotide comprises the nucleic acid sequence:
ATGGCTAGCGCTTCTCAGGTGCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCT
GGCGCCTCTGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCGGCTACTAC GTGCACTGGGTCCGACAGGCTCCAGGACAAGGACTGGAATGGATGGGCTGGGTCAA
CCCCAATAGCGGCGACACCAATTACGCCCAGAAATTCCAGGGCAGAGTGACCATGA
CCAGAGACACCAGCATCAGCACCGCCTACATGGAACTGAGCGGCCTGAGATCCGAT
GACACCGCCGTGTACTACTGCGCCAGAGATGGCGACGAGGACTGGTACTTCGATCTG
TGGGGCAGAGGCACCCCTGTGACAGTGTCTAGCGGAATCCTTGGATCTGGCGGCGGA
GGAAGCGGAGGCGGAGGTTCTGGTGGTGGCGGCTCTGACATTAGACTGACACAGAG
CCCTAGCAGCCTGAGCGCCAGCATCGGAGACAGAGTGACAATCACCTGTAGAGCCA
GCCAGGGCATCAGCAGATCCCTCGTGTGGTATCAGCAGAAGCCTGGCAAGGCCCCTC
GGCTGCTGATCTATGCTGCTAGCACACTGCAGAGCGGCGTGCCCTCTAGATTTTCTGG
CAGCGGCTCTGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCAGAGGACTT
CGCCACCTACTACTGCCTGCAGCACAACACATACCCCTTCACCTTCGGACCCGGCAC CAAGGTGGACATCAAGTCTGGAATCCCCGAGCAGAAGCTG (SEQ ID NO: 14).
[0123] In some embodiments, the second polynucleotide comprises the nucleic acid sequence: GAGATTCAGCTGCAGCAGTCTGGCCCCGAGCTTATGAAGCCTGGCGCCTCTGTGAAG
ATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCAGCTACTACATGCACTGGGTCAAG
CAGAGCCAGGGCAAGAGCCTGGAATGGATCGGCTTCATCGACCCCTTCAACGGCGG
CATCACCTACAACCAGAAGTTCAAGGGCAAAGCCACACTGACCGTGGACAGAAGCA
GCAGCACCGCCTATATGCACCTGAGAAGCCTGACCAGCGAGGACAGCGCCGTGTACT
ACTGCGCCAGATGCTACTACAACTACGACGACGAGGGCAGAGCCATGGACTATTGG
GGCCAGGGAACAAGCGTGACCGTGTCTAGTTCTGGCGGCGGAGGAAGCGGAGGCGG
AGGTTCTGGTGGTGGCGGCTCTGACATCCAGATGACACAGAGCCCCAGCAGCCTGTC
TGCCTCTCTGGGAGAAAGAGTGTCCCTGACCTGTAGAGCCAGCCAGGATATCGGCAG
CTCCCTGAATTGGCTGCAGCAGGGACCTGACGGCACCTTCAAGAGACTGATCTACGC
CACCTCCAGCCTGGATAGCAGCGTGCCCAAGAGATTCAGCGGCAGCAGAAGCGGCA
GCGACTACAGCCTGACAATCAGCTCTCTGGAAAGCGAGGACTTCGTGGACTACTACT
GCCTGCAGTACGCCAGCTCTCCCTACACATTTGGCGGAGGCACCAAGCTGGAAATCA AGCGG (SEQ ID NO: 29).
[0124] In some embodiments, the second polynucleotide comprises the nucleic acid sequence: ATGGCTGTTCTGGGCCTGCTGCTGTGCCTGGTCACCTTTCCAAGCTGTGTGCTGAGCC
AGGTGCAGCTGAAAGAGTCTGGACCTGGACTGGTGGCCCCTAGCCAGAGCCTGTCTA
TCACCTGTACCGTGTCCGGCTTCAGCCTGACCGATTACGGCGTGACCTGGATCAGAC
AGCCTCCTGGCAAAGGCCTGGAATGGCTGGGAGTTATTTGGGGCGGAGGCAGCACCT
ACTACAACAGCGCCCTGAAGTCCCGGCTGAGCATCAGCAAGGACAACAGCAAGTCC CAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATGTACTACTGT GCCAAGCACGAGAGCTACTACCCCTTCGTGTATTGGGGCCAGGGCACCCTGGTTACA GTGTCTGCTTCTGGCGGCGGAGGAAGCGGAGGCGGAGGTTCTGGTGGTGGCGGCTCT GACGTGGTCATGACACAGACACCTCTGAGCCTGCCTGTGTCTCTGGGAGATCAGGCC AGCATCAGCTGCAGAAGCAGCCAGAGCCTGATCAACAGCAACGGCAACACCTACCT GCACTGGTATCTGCAGAAACCCGGACAGAGCCCCAAGCTGCTGATCCACAGAGTGTC CAACAGATTCAGCGGCGTGCCCGACAGATTTTCTGGCAGCGGCTCTGGCACCGACTT CACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACCTGGGCGTGTACTTCTGTAGCCA GAGCACACACGTGCCCTGGACATTTGGCGGCGGAACAAAGCTGGAAATCAAGCGG (SEQ ID NO: 31).
[0125] In some embodiments, the second polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NOs: 6, 14, 29 or 31.
[0126] In some embodiments, the encoded VH comprises the amino acid sequence: QVKLQESGPGLVQPSQSLSFICTVSGFSLTSHGVHWVRQSPGKGLQWLGVIWGAGRTDY NAAFISRLSISRDISKSQVFFKMNSLQVDDTAIYYCARNRYESYFDYWGQGTTVTVSS (SEQ ID NO: 7).
[0127] In some embodiments, the encoded VH comprises the amino acid sequence: DVLMTNTPLSLPVSLGDQASISCRSSQNLVHSNGNTYLHWYLQKPGQSPNLLIYKVSNRF SGVPDRFSGSGSGTEFTLKISRVQAEDLGVYFCSQSTHVPLTFGAGSKLELK (SEQ ID NO: 9).
[0128] In some embodiments, the encoded VH comprises the amino acid sequence: EIQLQQSGPELMKPGASVKISCKASGYSFTSYYMHWVKQSQGKSLEWIGFIDPFNGGITY NQKFKGKATLTVDRSSSTAYMHLRSLTSEDSAVYYCARCYYNYDDEGRAMDYWGQGT SVTVSS (SEQ ID NO: 35).
[0129] In some embodiments, the encoded VH comprises the amino acid sequence: MAVLGLLLCLVTFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVTWIRQPPG KGLEWLGVIWGGGSTYYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAKHES YYPFVYWGQGTLVTVSA (SEQ ID NO: 39).
[0130] In some embodiments, the encoded VH comprises the amino acid sequence set forth in SEQ ID NOs: 7, 9, 35 or 39.
[0131] In some embodiments, the encoded VL comprises the amino acid sequence:
MASASQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVHWVRQAPGQGLEWMGWV NPNSGDTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDTAVYYCARDGDEDWYFDL WGRGTPVTVSSGIL (SEQ ID NO: 15).
[0132] In some embodiments, the encoded VL comprises the amino acid sequence: DIRLTQSPSSLSASIGDRVTITCRASQGISRSLVWYQQKPGKAPRLLIYAASTLQSGVPSRF SGSGSGDFTLTISSLQPEDFATYYCLQHNTYPFTFGPGTKVDIKSGIPEQKL (SEQ ID NO: 17).
[0133] In some embodiments, the encoded VL comprises the amino acid sequence: DIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQGPDGTFKRLIYATSSLDSSVPKR FSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPYTFGGGTKLEIKR (SEQ ID NO: 36).
[0134] In some embodiments, the encoded VL comprises the amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLINSNGNTYLHWYLQKPGQSPKLLIHRVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKR (SEQ ID NO: 40).
[0135] In some embodiments, the encoded VL comprises the amino acid sequence set forth in SEQ ID NOs: 14, 17, 36 or 40.
[0136] In some embodiments, the linker or the encoded linker comprises the amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 8).
[0137] In some embodiments, the linker or the encoded linker comprises the amino acid sequence: GSGGGGSGGGGSGGGGS (SEQ ID NO: 16).
[0138] In some embodiments, the linker or the encoded linker comprises the amino acid sequence: SGGGGSGGGGSGGGGS (SEQ ID NO: 42).
[0139] In some embodiments, the linker or the encoded linker comprises the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 16.
[0140] In some embodiments, the formed scFv comprises the amino acid sequence: QVKLQESGPGLVQPSQSLSFICTVSGFSLTSHGVHWVRQSPGKGLQWLGVIWGAGRTDY NAAFISRLSISRDISKSQVFFKMNSLQVDDTAIYYCARNRYESYFDYWGQGTTVTVSSGG GGSGGGGSGGGGSDVLMTNTPLSLPVSLGDQASISCRSSQNLVHSNGNTYLHWYLQKPG QSPNLLIYKVSNRFSGVPDRFSGSGSGTEFTLKISRVQAEDLGVYFCSQSTHVPLTFGAGS KLELK (SEQ ID NO: 10).
[0141] In some embodiments, the formed scFv comprises the amino acid sequence:
MASASQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVHWVRQAPGQGLEWMGWV NPNSGDTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDTAVYYCARDGDEDWYFDL WGRGTPVTVSSGILGSGGGGSGGGGSGGGGSDIRLTQSPSSLSASIGDRVTITCRASQGIS RSLVWYQQKPGKAPRLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQH NTYPFTFGPGTKVDIKSGIPEQKL (SEQ ID NO: 18).
[0142] In some embodiments, the formed scFv comprises the amino acid sequence: EIQLQQSGPELMKPGASVKISCKASGYSFTSYYMHWVKQSQGKSLEWIGFIDPFNGGITY NQKFKGKATLTVDRSSSTAYMHLRSLTSEDSAVYYCARCYYNYDDEGRAMDYWGQGT SVTVSSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQ GPDGTFKRLIYATSSLDSSVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPYTFGG GTKLEIKR (SEQ ID NO: 30).
[0143] In some embodiments, the formed scFv comprises the amino acid sequence: MAVLGLLLCLVTFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVTWIRQPPG KGLEWLGVIWGGGSTYYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAKHES YYPFVYWGQGTLVTVSASGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSS QSLINSNGNTYLHWYLQKPGQSPKLLIHRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED LGVYFCSQSTHVPWTFGGGTKLEIKR (SEQ ID NO: 32).
[0144] In some embodiments, the formed scFv comprises the amino acid sequence set forth in SEQ ID NO: 10, 18, 30, or 32.
[0145] In some embodiments, the encoded transmembrane domain comprises the amino acid sequence:
GDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIE MNRLGK (SEQ ID NO: 19).
[0146] In some embodiments, the encoded transmembrane domain comprises the amino acid sequence: AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 20).
[0147] In some embodiments, the encoded transmembrane domain comprises the amino acid sequence:
KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTV AFIIFWVRSKRSRLLHSDYM (SEQ ID NO: 21).
[0148] In some embodiments, the encoded transmembrane domain comprises the amino acid sequence: TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN RRRV (SEQ ID NO: 22).
[0149] In some embodiments, the encoded transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 19-22.
[0150] In some embodiments, any one of the first and second polynucleotides further comprises a nucleic acid sequence encoding a signal or a leading peptide.
[0151] In some embodiments, the encoded signal or leading peptide comprises the amino acid sequence: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 1).
[0152] In some embodiments, the encoded signal or leading peptide comprises the amino acid sequence: MKCLLYLAFLFIGVNCK (SEQ ID NO: 2).
[0153] In some embodiments, the encoded signal or leading peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
[0154] In some embodiments, any one of the first and second polynucleotides further comprises a promoter sequence.
[0155] In some embodiments, any one of the first to third nucleic acid sequences is operably linked to the promoter sequence.
[0156] In some embodiments, the polynucleotide further comprises at least one nucleic acid sequence encoding: a protein translation initiation motif, a tag, a cytoplasmic tail, a transcription termination motif, or any combination thereof.
[0157] In some embodiments, a protein translation initiation motif comprises a mammalian, e.g., a human, protein translation initiation motif.
[0158] Types of protein translation initiation motif would be apparent to one of ordinary skill in the art, including means for retrieving or obtaining their sequences. In some embodiments, a protein translation initiation motif comprises a Kozak consensus sequence or any functional equivalent thereof.
[0159] According to some embodiments, there is provided an expression vector comprising the polynucleotide(s) disclosed herein. In some embodiments, there is provided an expression vector comprising the first polynucleotide and the second polynucleotide. In some embodiments, the expression vector comprises a plasmid. [0160] In some embodiments, there is provided a viral vector. In some embodiments, the viral vector comprises a lentiviral vector. In some embodiments, the viral vector is expressed, at least partially, from the expression vector disclosed herein. In some embodiments, the lentiviral vector is expressed, at least in part, from expression vector disclosed herein. In some embodiments, the expression vector is or comprises a lentivirus-based expression vector.
[0161] According to some embodiments, there is provided a lentivirus comprising a mutated VSV- G protein as disclosed herein.
[0162] According to some embodiments, there is provided a lentivirus comprising an antibody or a fragment thereof having specific binding affinity to a hematopoietic cell surface marker.
[0163] According to some embodiments, there is provided a lentivirus comprising a mutated VSV- G protein as disclosed herein, and an antibody or a fragment thereof having specific binding affinity to a hematopoietic cell surface marker.
[0164] According to some embodiments, a hematopoietic cell is a HSPC a myeloid cell, a lymphoid cell, an erythrocyte, a mast cell, a myeloblast, T cell, B cell, NK cell, macrophage, monocyte, dendritic cell, neutrophil, or an eosinophil.
[0165] According to some embodiments, the antibody or a fragment thereof is a scFv.
[0166] According to some embodiments, there is provided a lentivirus comprising a scFv having specific binding affinity to a HSPC cell surface marker. Non-limiting examples for useful HSPC surface markers are CD34, CD90, CD117, CD133, CD59, and Flt3.
[0167] According to some embodiments, there is provided a lentivirus comprising a scFv having specific binding affinity to a T cell surface marker. Non-limiting examples for useful T surface markers are CD3, CD2, CD4, CD5, CD7, CD8, CD16, CD16a, CD28, CCR5, and CD107b.
[0168] According to some embodiments, there is provided a lentivirus comprising a scFv having specific binding affinity to a NK cell surface marker. Non-limiting examples for useful NK surface markers are CD56, CD244 and CD16.
[0169] According to some embodiments, there is provided a lentivirus comprising a scFv having specific binding affinity to a macrophage cell surface marker. Non-limiting examples for useful macrophage surface markers are CD14, CD16, CD64, CD 68 and CDl lb.
[0170] According to some embodiments, there is provided a lentivirus comprising a scFv having specific binding affinity to CD34. [0171] According to some embodiments, there is provided a lentivirus comprising a scFv having specific binding affinity to CD90 (also known as Thyl).
[0172] According to some embodiments, there is provided a lentivirus comprising: (i) a scFv having specific binding affinity to CD34; and (ii) a scFv having specific binding affinity to CD90.
[0173] According to some embodiments, there is provided a lentivirus comprising a bi-specific scFv having specific binding affinity to both CD34 and CD90.
[0174] In some embodiments, any one of the scFvs is integrated into the viral envelope of the lentivirus disclosed herein. In some embodiments, any one of the scFvs is integrated into the viral envelope via a transmembrane domain. In some embodiments, any one of the scFvs is integrated into the viral envelope via a transmembrane domain as disclosed herein.
[0175] In some embodiments, the lentivirus disclosed herein comprises a vesicular stomatitis virus G (VSV-G) protein. In some embodiments, the lentivirus disclosed herein comprises a viral envelope comprising a VSV-G protein.
[0176] In some embodiments, a VSV-G protein is or comprises a mutated VSV-G protein.
[0177] In some embodiments, a mutated VSV-G is characterized by having reduced or no binding affinity to a low-density lipoprotein (LDL) receptor.
[0178] In some embodiments, a mutated VSV-G protein maintains or preserves a pH-dependent membrane fusion.
[0179] In some embodiments, a mutated VSV-G protein comprises any VSV-G protein comprising at least one amino acid substitution, deletion, insertion, substitution or incorporation of non- naturally occurring amino acid, compared to the wildtype VSV-G protein, as long as the mutated VSV-G protein is: (i) characterized by having reduced or no binding affinity to a LDL receptor; (ii) maintains or preserves a pH-dependent membrane fusion; or (iii) both (i) and (ii).
[0180] In some embodiments, reduced or no binding affinity to an LDL receptor, is compared to a control VSV-G protein. In some embodiments, a control VSV-G protein comprises a wildtype VSV-G protein. In some embodiments, a control VSV-G protein comprises any mutated VSV-G protein comprising mutation(s) which do not affect and/or reduce and/or inhibit LDL receptor biding.
[0181] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) and an antibody or a fragment thereof having specific binding affinity to a hematopoietic cell surface marker. [0182] According to some embodiments, the antibody or a fragment thereof is a scFv.
[0183] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD34
[0184] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD90.
[0185] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD3, CD4, CD8 or any combination thereof.
[0186] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) a scFv having specific binding affinity to CD34; and (iii) a scFv having specific binding affinity to CD90.
[0187] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) a scFv having specific binding affinity to CD3; and (iii) a scFv having specific binding affinity to CD4.
[0188] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) a scFv having specific binding affinity to CD3; and (iii) a scFv having specific binding affinity to CD8.
[0189] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) a bi-specific scFv having specific binding affinity to both CD34 and CD90.
[0190] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD3.
[0191] According to some embodiments, there is provided a lentivirus comprising a (i) mutated VSV-G protein and (ii) and scFv having specific binding affinity to CD56
[0192] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light/heavy chain pair form an antibody binding site. An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelid, CDR- grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitopebinding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences. An antibody may be from any species. The term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulfide-linked variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. The skilled artisan will further appreciate that other fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH)~ Fc fusions and scFv-scFv-Fc fusions.
[0193] In some embodiments, a lentivirus-based vector comprises an inactivated lentiviral vector. In some embodiments, a lentivirus-based vector comprises a third-generation lentiviral vector. In some embodiments, a lentivirus-based vector comprises a self-inactivating lentiviral vector. In some embodiments, a lentivirus-based vector is devoid of a trans-activator of transcription encoding gene (Tat). In some embodiments, a lentivirus-based vector is devoid of one or more viral accessory proteins. In some embodiments, a viral accessory protein is selected from: vif, vpr, vpu, nef, or any combination thereof. In some embodiments, a lentivirus-based vector comprises any lentivirus-based vector suitable for human therapy. In some embodiments, suitable comprises safe for human health.
Cells
[0194] According to some embodiments, there is provided a cell comprising: (a) any one of the polynucleotides disclosed herein; (b) the expression vector disclosed herein; (c) the lentivirus disclosed herein; or (d) any combination of (a) to (c).
[0195] In some embodiments, the cell is a transduced cell.
[0196] In some embodiments, transduced comprises being transduced with the expression vector of the invention.
[0197] In some embodiments, the cell is an animal cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell or a human cell line.
[0198] In some embodiments, the cell is configured to or optimized for expression, production, packing, secretion, assembly, or any combination thereof, of the lentivirus disclosed herein. [0199] In some embodiments, the cell is suitable for expression, production, packing, secretion, assembly, or any combination thereof, of the lentivirus disclosed herein.
[0200] In some embodiments, the cell is used in a method for expression, production, packing, secretion, assembly, or any combination thereof, of the lentivirus disclosed herein.
[0201] According to another aspect, there is provided a method for expressing, producing, packing, secreting, assembling, or any combination thereof, of the lentivirus disclosed herein.
[0202] In some embodiments, the method comprises culturing the cell as disclosed herein under conditions sufficient for the cell to express the expression vector disclosed herein, thereby expressing, producing, packing, secreting, assembling, or any combination thereof, of the lentivirus disclosed herein.
Compositions
[0203] According to some embodiments, there is provided a composition comprising: (a) the polynucleotides disclosed herein; (b) the expression vector disclosed herein; (c) the lentivirus disclosed herein; (d) the cell disclosed herein, or (e) any combination of (a) to (d).
[0204] In some embodiments, the composition further comprises an acceptable carrier, adjuvant, or excipient. In some embodiments, the carrier, adjuvant, or excipient is a pharmaceutically acceptable carrier, adjuvant, or excipient.
[0205] In some embodiments, the composition is for use in in-vivo gene therapy in a subject in need thereof.
[0206] In some embodiments, the subject is afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor cell thereof.
[0207] In some embodiments, the composition is for use in transfection or transduction, or introduction of an exogenous nucleic acid molecule into a subject in need thereof.
[0208] In some embodiments, in-vivo gene therapy comprises transfection or transduction, or introduction of an exogenous nucleic acid molecule into a subject in need thereof.
Methods of use and preparation
[0209] According to another aspect, there is provided a method for preparing an improved lentivirus targeting a hematopoietic cell for in vivo gene therapy, the method comprising: contacting a cell with: (a) a first expression vector comprising a nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G; and (b) a second expression vector comprising a nucleic acid sequence comprising: a nucleic acid sequence encoding an antibody heavy chain variable region (VH); a nucleic acid sequence encoding an antibody light chain variable region (VL); and a nucleic acid sequence encoding a transmembrane domain, wherein the encoded VH and VL form a single-chain variable fragment (scFv), and culturing the cell under conditions such that the nucleic acid sequence of the first expression vector and the nucleic acid sequence of the second expression vector are expressed, thereby, preparing the improved lentivirus targeting a hematopoietic cell for in vivo gene therapy.
[0210] In some embodiments, the contacting is at a copy number wherein the copy number of the second expression vector is equal to or greater than the copy number of the first expression vector. In some embodiments, the copy number of the second expression vector supersedes the copy number of the first expression vector. In some embodiments, the copy number of the second expression vector is greater than the copy number of the first expression vector. In some embodiments, the copy number of the first expression vector is lower than or equal to the copy number of the second expression vector.
[0211] In some embodiments, the contacting is at a copy number ratio ranging between 0.01:1 and 1:1, 0.02:1 and 1:1, 0.05:1 and 1:1, 0.08:1 and 1:1, 0.1:1 and 1:1, 0.2:1 and 1:1, 0.3:1 and 1:1, 0.5:1 and 1:1, 0.7:1 and 1:1, and 0.9:1 and 1:1 of the first expression vector and the second expression vector. Each possibility represents a separate embodiment of the invention.
[0212] In some embodiments, an improved lentivirus is characterized by: reduced resistance onset, increased stability, reduced serum inactivation, increased titer, increased infectivity or any combination thereof, in a subject administered therewith, compared to a control lentivirus.
[0213] According to another aspect, there is provided a method for improving in vivo gene therapy targeting a hematopoietic cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lentivirus comprising: (a) a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) positions 47-50; (iii) positions 184-189; (iv) positions 349-353; and (v) any combination of (i) to (iv), compared to a wildtype VSV-G; and (b) a second nucleic acid sequence comprising a sequence encoding an antibody VH; a sequence encoding an antibody VL; and a sequence encoding a transmembrane domain, wherein the encoded VH and VL form a scFv, wherein the lentivirus is obtained or derived from a cell comprising the first nucleic acid sequence and the second nucleic acid sequence at a copy number ratio of ranging between 0.01:1 and 1:1.
[0214] In some embodiments, the method further comprises a step preceding the administering, comprising preparing the lentivirus as disclosed herein.
[0215] In some embodiments, improving comprises: reducing resistance of the subject to the lentivirus, increasing abundance and/or stability of the lentivirus in the subject, reducing inactivation (e.g., proteolytic digestion) of the lentivirus in serum of the subject, or any combination thereof, compared to a control lentivirus.
[0216] In some embodiments, a control lentivirus is not the lentivirus of the invention. In some embodiments, a control lentivirus comprises a WT VSV-G. In some embodiments, a control lentivirus comprises a mutated VSV-G being devoid of the mutation(s) disclosed herein. In some embodiments, a control lentivirus is devoid of a scFv. In some embodiments, a control lentivirus is devoid of a scFv as disclosed herein.
[0217] According to some embodiments, there is provided a method for introducing a nucleic acid molecule of interest predominantly or specifically into a hematopoietic cell or a progenitor thereof in a subject in need thereof.
[0218] According to some embodiments, there is provided a method for determining the suitability of a subject for in vivo hematopoietic gene therapy treatment.
[0219] In some embodiments, the method comprises the steps of: (a) providing a blood sample obtained or derived from the subject; (b) contacting the blood sample with a vector as disclosed herein; (c) determining the rate of vector inactivation in the blood sample of step (b), wherein a rate of vector inactivation being below a predetermined threshold is indicative of the subject being suitable for in vivo hematopoietic gene therapy treatment, thereby for determining the suitability of a subject for in vivo hematopoietic gene therapy treatment.
[0220] In some embodiments, a rate of vector inactivation being equal to or above a predetermined threshold is indicative of the subject being unsuitable for in vivo hematopoietic gene therapy treatment, as disclosed herein.
[0221] According to some embodiments, there is provided a method for treating a subject afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor cell thereof.
[0222] In some embodiments, step (a) of the method disclosed herein comprises obtaining a blood sample from a subject. [0223] In some embodiments, the method comprises administering to the subject an effective amount of: (a) the expression vector disclosed herein; (b) the lentivirus disclosed herein; or (c) the composition disclosed herein; and a nucleic acid molecule of interest, thereby introducing a nucleic acid molecule of interest predominantly or specifically into a hematopoietic cell or a progenitor thereof of the subject.
[0224] In some embodiments, the therapeutic nucleic acid molecule is configured to substituting or rectifying a genetic loss of function of a blood cell or a progenitor cell thereof, thereby treating the subject afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor cell thereof.
[0225] In some embodiments, the therapeutic nucleic acid molecule is configured to introducing a therapeutic gene product to a blood cell or a progenitor cell thereof, thereby treating a subject afflicted with a genetic disease.
[0226] In some embodiments, introducing is into the genome of a hematopoietic cell or a progenitor thereof, in the subject.
[0227] In some embodiments, the hematopoietic cell or the progenitor cell is characterized by CD34 expression, CD90 expression, or both.
[0228] In some embodiments, expression comprises presence or increased abundance of an mRNA transcript of the CD34 gene, the CD90 gene, or both, presence or increased abundance of a protein product translated therefrom, or any combination thereof.
[0229] In some embodiments, a hematopoietic cell, comprises a stem cell. In some embodiments, a stem cell is a hematopoietic stem cell. In some embodiments, a hematopoietic cell, comprises a T cell or a NK cell.
[0230] The term "stem cell" is used herein to refer to a cell (e.g., a vertebrate stem cell) that has the ability both to self-renew and to generate a differentiated cell type (see Morrison et al. (1997) Cell 88:287-298). Stem cells may be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny.
[0231] As used herein, the terms "hematopoietic stem cell" or "HSC" refer to an undifferentiated hematopoietic cell that is capable of differentiating into all blood cell types, myeloid and lymphoid cells. The HSC may reside in the bone marrow or be found elsewhere e.g., peripheral blood. [0232] In some embodiments, the hematopoietic cell or the progenitor cell comprises at least one genetic aberration conferring a disease.
[0233] In some embodiments, the genetic aberration comprises a mutation, deletion, insertion, inversion, translocation, or any combination thereof.
[0234] In some embodiments, the hematopoietic cell or the progenitor cell comprises at least one loss of function mutation. In some embodiments, the mutation is inducing, resulting, or conferring a disease. In some embodiments, the loss of function mutation comprises a nonsense mutation or missense mutation.
[0235] In some embodiments, predominantly or specifically is at least by 2-fold, 3-fold, 5-fold, 10- fold, 20-fold, 30-fold, 50-fold, 70-fold, 100-fold, 250-fold, 350-fold, 500-fold, 750-fold, or 1,000- fold, compared to control, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.
[0236] In some embodiments, a control comprises a non-hematopoietic cell or any progenitor thereof. In some embodiments, a control comprises a cell devoid of expression or characterized by under-expression or downregulation of CD34, CD90, or both. In some embodiments, a control comprises a cell having a cell surface devoid of CD34, CD90, or both.
[0237] In some embodiments, administering comprises intravenously administering.
[0238] In some embodiments, administering comprises locally administering.
[0239] In some embodiments, administering comprises intravenously administering and locally administering.
[0240] In some embodiments, administering comprises intraosseous infusion.
[0241] In some embodiments, administering comprises intrathymically administering.
[0242] In some embodiments, administering comprises intrathecally administering.
[0243] In some embodiments, transducing and/or introducing comprises transferring an expression vector comprising the polynucleotide molecule into a target cell.
[0244] In some embodiments, a target cell comprises or is a hematopoietic cell or a progenitor thereof.
[0245] In some embodiments, transferring is or comprises transfecting. In some embodiments, transferring is or comprises lipofecting. In some embodiments, transferring is or comprises nucleofecting. In some embodiments, transferring is or comprises viral infection. [0246] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.
[0247] As used herein, the terms “administering”, “administration” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.
[0248] As used herein, the terms “subject” or “individual” or “animal” or “patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.
General
[0249] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.
[0250] It is noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only", and the like in connection with the recitation of claim elements or use of a "negative" limitation.
[0251] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."
[0252] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
[0253] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
EXAMPLES
[0254] Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples. Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.
[0255] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include chemical, molecular, biochemical, and cell biology techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); The Organic Chemistry of Biological Pathways by John McMurry and Tadhg Begley (Roberts and Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions by Richard Silverman (Academic Press, 2002); Organic Chemistry (6th Edition) by Leroy "Skip" G Wade; Organic Chemistry by T. W. Graham Solomons and, Craig Fryhle. EXAMPLE 1
Construction of membrane-bound scFv for HSC targeting and optimal TM Domain selection
[0256] To express the CD34 or CD90 scFv on the 293T cells, different signal peptide (Table 1) and transmembrane domains (Table 2) combinations were tested for efficient expression on the 293T cell membrane. First, cloning of the expression plasmids was generated based on the scheme depicted in Fig. 1. Cloning was executed by combining different molecular biology methods including PCR, gene synthesis and Gibson assembly. The plasmids containing the desired scFv (e.g., anti-CD34 clones 4C8 ,2E10, 5B12, anti CD3, anti MPL, anti C-Kit clones 79D, CK6 and anti CD90, Table 3) were transfected into 293T cells. Twenty-four (24) hours later, cells were collected and analyzed for transmembrane expression of the scFv for both positive cells and intensity using the anti-myc tag. GFP plasmid was used to normalize transfection rates. Transfection efficiency was even in all tested constructs as determined by GFP expression (data not shown). The highest Myc expression was observed in constructs #7 and #8 harboring the CD8 transmembrane domain and the signal peptide of the murine Ig K chain and VSVG, respectively (Fig. 2A). Myc expression of the different scFvs tested with the optimal CD8 transmembrane domain and the signal peptide of VSVG configuration (Fig. 2B).
Table 1. Signal peptides sequences
Figure imgf000037_0001
Table 2. Transmembrane domain table (Extracellular region - transmembrane - cytoplasmic tale)
Figure imgf000037_0002
Figure imgf000038_0001
Table 3. scFv nucleotide and amino acid sequences
Figure imgf000038_0002
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0002
EXAMPLE 2
Identification of potential LDL-R binding VSV-G residues
[0257] The protein sequences of all glycoproteins were downloaded from uniport, accession numbers P03522, 056677, F8SPF4, Q85213 and P1318O respectively. Alignment was performed using the Snapgene software adopting the MUSCLE algorithm (Fig. 3). Potential residues for substitution within the important CR2-CR3 binding segments (excluding residues previously identified mutations by Nikolic et al.) that were identified based on this alignment are shown in table 4.
Table 4. Potential mutant residues (Position related to proteins w/o a signal peptide)
Figure imgf000044_0001
Figure imgf000045_0001
EXAMPLE 3
Comparison of infection capacity of selected mutants to wt VSV-G
[0258] To test the infectivity of mutants H49A, K50T, T352A and E353P, viruses containing wt or mut VSV-G were produced. Briefly, 293T cells were co-transfected with the lentiviral vector pCCL-PGK-GFP, plasmids encoding viral gag, pol, and rev genes and the wt or mutVSV-G. For transfection, the TransIT-VirusGEN® Transfection Reagent (Minis) was used. Generated LVs were collected and filtered 48 hours after transfection. LVs titers were determined using the Lenti- X p24 Rapid Titer Kit (Takara Bio cat. 632200). VSV-G mutant titers were approximately 0.7-fold of wt VSV-G. Transduction of 293T or U937 cells was carried out by mixing the viruses with polybrebe (Sigma) transduction enhancer. For human primary CD34+ and activated T cells, transduction was performed in the presence of the LentiBOOST (sirion biotech) transduction enhancer. Twenty-four (24) hours later, cells were washed to remove any virions left in the media. Cells were maintained and collected for analysis 4-5 days post transduction. To evaluate co- exitance of binding and fusion events of the transfected cells, percentage of positive GFP cells were measured using flow cytometry. No reduction in infectivity was measured in mutants H49A and E353P. In contrast, reduction in percentage of positive GFP cells was measured when K50T and T352A mutants were tested. This reduction was further enhanced when a double mutant (K50T and T352A) LV was tested (Fig. 4A and 4B). Ultimately, in comparison to wt VSV-G, the double mutant K50T and T352A demonstrated an average of 50- to 100-fold reduction of infectivity in human primary CD34+ and activated T cells respectively (Fig. 4C).
EXAMPLE 4
Analyzing expression of VSV-G mutants on 293T cells membrane
[0259] To verify that the observed reduced binding capacity is not a result of low membrane expression, K50T and T352A mutants and wt VSV-G plasmids were transfected with equal DNA amounts to 293T cells. Twenty-four (24) hours post transfection, cells were collected and stained with anti- VSV-G (Abeam ab3863) to measure VSV-G expression levels by flow cytometry (Fig. 5).
EXAMPLE 5
Optimizing scFv plasmid and VSV-G concentration
[0260] To maximize VSV-G and scFv co-expression on the same virion, the levels of scFv were calibrated by co-transfection of both the scFv and VSV-G plasmids into 293T LV production cell line. Twenty-four (24) hours post transfection, cells were incubated with anti-VSVG and anti-myc tag and number of double positive 293T transfected cells was measured by flow cytometry analysis.
[0261] VSV-G plasmid concentration was 100 ng/pl and tested scFv concentrations were set to 200 ng/pl ,100 ng/pl, 50 ng/pl and 25 ng/pl corresponding to 1:2, 1:1, 2:1 and 4:1 VSV-G to scFv ratios. The scFvs included in this set of experiments were anti-CD90 and anti-CD34 5B 12 and 2E10 clones, respectively. Among all the tested ratios, the 1:1 and 2:1 VSV-G to scFv ratios demonstrated the best co-expression levels (Fig. 6). Therefore, the 1:1 ratio (100 ng/pl of each plasmid) was chosen for further investigation and LV production.
[0262] Production of LV-scFv -VSVG K50T T352A was achieved by co-transfection of 293T cells with a lentiviral vector pCCL-PGK-GFP, plasmids encoding viral gag, pol, and rev genes and the VSV-G K50T T352A in combination with scFv plasmids. As a control, the Env derived from VSVGWT was used as a joint recognition and fusion protein. For transfection, the TransIT-VirusGEN® Transfection Reagent (Minis) was used and the generated viruses were collected, filtered and centrifuge 48 hours after transfection.
EXAMPLE 6
Incorporation and transduction efficacy to cell lines
[0263] To test for scFv -VSVG K50T T352A LV specificity, 293T stable cell lines were prepared. Briefly, 293T cells were transduced with CD90+_puromycin, CD34+_puromycin or MPL+_puromycin LVs in the presence of polybrene transfection enhancer. Twenty-four (24) hours later, cells were washed to remove any virion debris. Cells were maintained in puromycin free DMEM for 3 days to recover and then incubated in DMEM containing puromycin for selection. To confirm CD90+/CD34+/MPL+ expression, samples from the stable cell lines were collected and incubated with antibodies against CD90, CD34 and MPL respectively. Flow cytometry was used to demonstrate successful markers expression (Fig. 7A-7C). The generated cell lines were transduced with LV- scFv4C8-VSVG K50T-T352A , LV scFvCD90-VSVG K50T-T352A or LV- scFvMPL-VSVG K50T T352A . For negative control, the parental cell lines were used. To test for binding events efficacy in the presence or absence of the scFv/VSV-GK50T T352A , VSVG K50T-T352A as well as scFv LVs were used as controls (data not shown). In addition, LV-VSVGWT was used as a reference for efficient transduction. Transduction was carried out by mixing the viruses with polybrene (Sigma) transduction enhancers into an appropriate tissue culture dish. The cells, in which puromycin was removed for transduction, were added to the well at a concentration of 0.5- IxlO6 cells/ml. Twenty-four (24) hours later, cells were washed to remove any virions left in the media. Cells were maintained and collected for analysis 5 days post transduction.
[0264] To evaluate co-exitance of binding and fusion events of the transfected cells, percentage of positive GFP cells were measured using flow cytometry. 293T_CD90- cells transduced with SCFVCD90-VSVG K50T T352A LV, demonstrated a reduction in infectivity levels of approximately 5- fold, similarly to the reduction that was measured in 293T cell transduced with VSVG K50T-T352A LV. However, in 293T_CD90+ cells, a dramatic increase in infectivity level was measured. This increase was comparable to cells transduced with VSVG wt LV (1.16-fold, Fig. 8A). Likewise, in 293T_CD34+ cells, infectivity level of cells transduced with scFv4C8-VSVG K50T-T352A and scFv5B12-VSVG K50T T352A LVs were 0.97 and 0.2-fold in comparison to cells transduced with VSVG wt LV (Fig. 8B). To further test for specificity, 293T+CD34 cells were co-cultured and transfected with either VSVG wt or scFv4C8-VSVG K50T-T352A LVs. While transfection rates between the two cell populations were similar when VSVG wt LV was used (1.4-fold change), meaningful difference was measured when scFv4C8-VSVGK50T T352ALV was transduced (21-fold change, Fig. 8C). Transduction with scFvCD3-VSVGK50T T352ALV and scFvL61-VSVGK50T T352A LV resulted in 0.97 and 0.66 infectivity levels in comparison to VSVG WT LV in Jurkat and 293T_MPL+ cells respectively (Fig. 8D and 8E). These results clearly show that while the VSVG K5OT_T352A as rcc|Llccc| capacity to bind the LDL-R, it maintains its membrane fusion capacity. The addition of the scFv to the envelope virion allows to regain high infectivity levels by compensating for the loss of binding of the K50T_T352A mutant.
[0265] Modifications on the virus envelope are known to impact LV titers. To test the effect of addition and the mutants the scFv, functional titers were measured. No dramatic reduction in transduction units (TU) /ml were observed suggesting feasible production for in-vivo delivery (Table 5).
Table 5. Functional titers of VSVG WT , scFvCD90 VSVG K50T-T352A and scFv4C8VSVG
K50T_T352A
Figure imgf000048_0001
EXAMPLE 7
Incorporation and transduction efficacy of primary human cells
[0266] To evaluate the ability of the scFv -VSVG K50T T352A LV to transduce primary human cells, mobilized peripheral blood cells (MPBCs) derived CD34+ human hematopoietic stem cells (HSCs) and peripheral blood mononuclear cells (PBMC) derived CD3+ human T cells were used. Granulocyte colony stimulating factor (G-CSF) MPBCs were collected by apheresis from healthy donors at the Ezer Mizion Bone Marrow Collection Site. CD34+ cells are purified by CD34 positive selection microbeads (Miltenyi). T cells were purified using the human Pan T cell isolation kit (Millteny). Cell purity was measured using flow cytometry (93% CD34+ and 98% CD3+ cells; data not shown).
[0267] T cells were activated using TransAct (Millteny) according to the manufacturer's instructions. For transduction, resting and activated human T cells were cultured in LymphoONE T cells expansion media (Takara) supplemented with IL-2 (PeproTech) and human serum (sigma) for 48h prior to transduction, human G-CSF MPBC-derived CD34+ cells were cultured in serum- free SCGM medium (CellGenix) supplemented with a cocktail of cytokines: IL-3, TPO, SCF, and FLT-3L (PeproTech) for minimal incubation prior to transduction. Transduction was carried out by mixing the viruses with the LentiBOOST (Sirion) transduction enhancer into an appropriate tissue culture dish. The CD3+ or CD34+ cells were added to the well at a final concentration of 0.9xl06 cells/ml. Twenty-four (24) hours later, cells were washed to remove any virions left in the media. Cells were maintained and collected for analysis 4 days post transduction. Transduced cells were evaluated by FACS with the following markers: CD34+, CD3+, and GFP. To demonstrate specificity in CD34+, cells were gated based on CD34 level (Fig. 9A). While no difference was observed in both population infectivity rates when VSVG WT LV was used, a clear and significant difference of 71-fold was measured in cells transduced with scFv- VSVG K50T-T352A LV (Fig 9B). In a similar manner, scFvCD90-VSVG K50T -T352A, scFvMPL-VSVG K50T-T352A and scFvC-KIT- VSVG K50T T352A LVs were tested with no GFP expression measured either due to low level of marker expression (CD90 and C-KIT) or low binding avidity of the scFv (MPL, data not shown). In activated T cells transduced with VSVG WT LV, infectivity level was high with about 85% transduction rate. As expected, and previously reported (Amirache et al., 2014), very low transduction rates were measured in resting T cells. In contrast, when Anti-CD3- VSVG K50T-T352A LV was used, high transduction rates were measured in both activated and resting T cells. The successful transduction of resting T cells, supports the argument that Anti-CD3- VSVG K50T-T352A LV enter the cell through CD3 and not LDL-R.
EXAMPLE 8 scFv -VSVG K50T-T352A j y ()|p jarget jn hepatocytes
[0268] In vivo VSVG WT LV injection results in high transduction rate in hepatocytes, partly due to the high level of LDL-R on these cells (Milani et al., 2022; 2019). The inventors hypothesized that transduction with scFv -VSVG K50T T352A LV would potentially reduce transduction rates in comparison to VSVG WT LV, resulting in decreasing liver off-target effect in in vivo setting. To test this, HUH7 and HepG2 hepatocytes cell lines were transduced with either VSVG WT LV or 4C8 - VSVGK50T T352A LV in the presence of polybrene (Sigma) transduction enhancer. Twenty-four (24) hours later, cells were washed to remove any virions left in the media. Cells were maintained and collected for analysis 3 days post transduction. Approximately 5-fold reduction in infection rate was measured when the 4C8 -VSVG K50T-T352A LV in comparison to VSVG WT LV (Fig 10A). Transduction of mouse primary hepatocytes resulted in GFP positive signal measured by fluorescence microscopy in cells transduced with VSVG WT LV 5 days post transduction. On the contrary, no GFP signal was observed when cells were transduced with 4C8 -VSVGK50T T352A LV (Fig 10B). These results suggest an advantage of the scFv -VSVGK50T T352A LVs in terms of safety and efficacy aspects in the context of in vivo delivery.
EXAMPLE 9
Serum sensitivity of LVs with VSV-G or Cocal glycoproteins
[0269] VSV-G and Cocal resistance to complement-mediated inactivation was tested by preincubation of five-fold diluted VSV-G and Cocal pseudotyped LVs in fresh or heat inactivated (HI) human serum for 1 hour at 37 °C (QUIDEL, Al 13). Following virus-serum incubation, 293 cells were infected at multiplicity of infection 1 (MOI1) and viral infectivity was measured by flow cytometry 3 days post transduction. Infectivity levels post incubation in serum were dramatically reduced by 7.6- and 6.7-fold using VSV-G and Cocal LVs, respectively (Fig. 11A). As opposed to previous publications, we did not observe a substantial difference in serum sensitivity between of VSV-G and the Cocal pseudotyped LVs.
EXAMPLE 10
Lower serum sensitivity of VSV-G -LVs with reduced amounts of VSV-G
[0270] Decreasing amounts of VSV-G expression plasmid (100, 50, and 25 ng/pl VSV-G, Fig. 11B) were used to produce scFvCD90_VSV-GWTLVs as described above. LV with decreasing VSV-G content were then incubated in human sera as described in the previous section. Titer recovery as measured by infectivity showed increased resistance to inactivation in human sera in correlation with reduced VSV-G levels (Fig. 11C).
EXAMPLE 11
The scFv-non-LDL-R binding LV maintains high infectivity levels at lower VSV-G concentrations in comparison to VSVG WT LV
[0271] Decreasing amounts of wt or T352A VSV-G expression plasmid (100, 50, and 25 ng/pl) were used to produce LVs expressing scFv targeted against the CD90 marker. Next, LVs were transduced into CD90 positive or negative 293T cells. When scFv _VSV-G WT LVs are used to transduce 293T CD90’ cells, binding solely depends on the LDL-R. However, when 293T CD90+ cells are transduced, binding can occur either from the LDL-R or the CD90 marker. Similarly, utilizing the VSV-GT352A, binding to CD90+ is mediated through the CD90 marker. To complete infection, VSV-G is solely required for membrane fusion and not binding, potentially demanding lower levels of expression in comparison to when both binding and fusion are VSV-G dependent. Indeed, infectivity depended on VSV-G levels were better maintained in LVs expressing the VSV- GT352A, probably due to its ability to bind the cell through the scFv targeted against CD90 and not through the VSV-G mediated LDL-R binding (Fig. 12).
EXAMPLE 12
The K50T and T352A increase VSV-G serum resistance levels
[0272] At 50 ng/pl, VSV-G concentration reduced serum sensitivity was measured, while infectivity levels were still maintained (Fig. 12). Therefore, this concentration was determined as the optimal level for further investigation. For determination of the effect of this concentration on serum inactivation, scFvCD90 _VSV-G T352A and VSV-G WT LVs were tested for human serum inactivation by incubating the virions with human serum (QUIDEL, Al 13) at 37 °C for 1 hr. Next, the incubated virions were transduced into 293T_ CD90+ cells and GFP levels were measured by flow cytometry to determine infection level. The scFvCD90_VSV-G T352A was 2-fold more resistance to human serum in comparison to VSV-G WTLVs (Fig. 13).
[0273] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS What is claimed is:
1. A method for preparing an improved lentivirus targeting a hematopoietic cell for in vivo gene therapy, the method comprising: contacting a cell with: a. a first expression vector comprising a nucleic acid sequence encoding a mutated vesicular stomatitis virus G protein (VSV-G), the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G; and b. a second expression vector comprising a nucleic acid sequence comprising: i. a nucleic acid sequence encoding an antibody heavy chain variable region (VH); ii. a nucleic acid sequence encoding an antibody light chain variable region (VL); and iii. a nucleic acid sequence encoding a transmembrane domain, wherein said encoded VH and VL form a single-chain variable fragment (scFv), wherein the contacting is at a copy number ratio ranging between 0.01:1 and 1:1 of said first expression vector and said second expression vector; and culturing said cell under conditions such that said nucleic acid sequence of said first expression vector and said nucleic acid sequence of said second expression vector are expressed, thereby, preparing the improved lentivirus targeting a hematopoietic cell for in vivo gene therapy.
2. The method of claim 1, wherein said improved lentivirus is characterized by: reduced resistance onset, increased stability, reduced serum inactivation, increased titer, increased infectivity or any combination thereof, in a subject administered therewith, compared to a control lentivirus.
3. A method for improving in vivo gene therapy targeting a hematopoietic cell in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a lentivirus comprising: (a) a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8- 10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV- G; and (b) a second nucleic acid sequence comprising a sequence encoding an antibody VH; a sequence encoding an antibody VL; and a sequence encoding a transmembrane domain, wherein said encoded VH and VL form a scFv, wherein said lentivirus is obtained or derived from a cell comprising said first nucleic acid sequence and said second nucleic acid sequence at a copy number ratio of ranging between 0.01:1 and 1:1.
4. The method of claim 3, wherein said improving comprises: reducing resistance of said subject to said lentivirus, increasing stability of said lentivirus in said subject, reducing inactivation of said lentivirus in serum of said subject, or any combination thereof, compared to a control lentivirus.
5. The method of any one of claims 1 to 4, wherein said administering is via a route selected from the group consisting of: intravenous, local, intraosseous infusion, intrathymical, intrathecal, and any combination thereof.
6. The method of any one of claims 1 to 5, wherein said mutated VSV-G further comprises at least one amino acid substitution of positions 47-50, compared to said wildtype VSV-G.
7. The method of any one of claims 1 to 6, wherein said mutated VSV-G comprises: (i) a substitution of lysine at position 50 (K50) to threonine; (ii) a substitution of threonine at position 352 (T352) to alanine; or (iii) both (i) and (ii).
8. The method of any one of claims 1 to 7, wherein said mutated VSV-G is characterized by having reduced or no binding affinity to a low-density lipoprotein (LDL) receptor, compared to a control VSV-G.
9. The method of any one of claims 1 to 8, wherein said scFv is characterized by having specific binding affinity to any one of cluster of differentiation CD 34, CD90, and both.
10. The method of any one of claims 1 to 9, wherein said nucleic acid sequence encoding said antibody VH comprises the nucleic acid sequence set forth in SEQ ID Nos: 3, 33, 37, or 11.
11. The method of any one of claims 1 to 10, wherein said nucleic acid sequence encoding said antibody VL comprises the nucleic acid sequence set forth in SEQ ID Nos: 5, 34, 38, or 13.
12. The method of any one of claims 1 to 11, wherein said lentivirus comprises the nucleic acid sequence set forth in SEQ ID Nos: 6, 29, 31 or 14.
13. The method of any one of claims 1 to 12, wherein the encoded VH comprises the amino acid sequence set forth in SEQ ID Nos: 7, 35, 39, or 9.
14. The method of any one of claims 1 to 13, wherein the encoded VL comprises the amino acid sequence set forth in SEQ ID Nos: 15, 36, 40 orl7.
15. The method of any one of claims 1 to 14, wherein said formed scFv comprises the amino acid sequence set forth in SEQ ID Nos: 10, 30, 32 or 18.
16. The method of any one of claims 1 to 15, wherein said encoded transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 19-22.
17. The method of any one of claims 1 to 16, wherein said subject is afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor thereof.
18. A synthetic polynucleotide comprising a first nucleic acid sequence encoding a mutated VSV-G, the mutated VSV-G comprising at least one amino acid substitution in any one of: (i) positions 8-10; (ii) 184-189; (iii) 349-353; and (iv) any combination of (i) to (iii), compared to a wildtype VSV-G.
19. The synthetic polynucleotide of claim 18, further comprising a second nucleic acid sequence comprising: a. a nucleic acid sequence encoding an antibody VH; b. a nucleic acid sequence encoding an antibody VL; and c. a nucleic acid sequence encoding a transmembrane domain, wherein said encoded VH and VL form a scFv.
20. An expression vector comprising the synthetic polynucleotide of claim 18 or 19.
21. The expression vector of claim 20, being a lentivirus-based expression vector.
22. A viral vector comprising the expression vector of claim 20 or 21;
23. The viral vector of claim 22, being a lentivirus.
24. The lentivirus of claim 23, wherein said scFv is integrated into a viral envelope of said lentivirus via said transmembrane domain.
25. A cell comprising: a. the synthetic polynucleotide of claim 18 or 19; b. the expression vector of claim 20 or 21 ; c. the viral vector of claim 22 or 23; d. the lentivirus of claim 24; or e. any combination of (a) to (d).
26. A composition comprising: a. the synthetic polynucleotide of claim 18 or 19; b. the expression vector of claim 20 or 21 ; c. the viral vector of claim 22 or 23; d. the lentivirus of claim 24; e. the cell of claim 25; or a. any combination of (a) to (e); and an acceptable carrier.
27. The composition of claim 26, wherein said carrier is a pharmaceutically acceptable carrier.
28. The composition of claim 27, for use in in vivo gene therapy in a subject in need thereof.
29. The composition of claim 28, wherein subject is afflicted with a disease comprising genetic loss of function of a blood cell or a progenitor thereof.
PCT/IL2023/050234 2022-03-06 2023-03-06 Lentiviral vectors for in vivo targeting of immune cells WO2023170681A1 (en)

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