Chimeric VSV-G proteins as nucleic acid transfer vehicles

Abstract

The design and generation of a number of chimeric VSV-G (or VSV-G variants) proteins are used as transfer vehicles to enhance delivery of nucleic acids like plasmid DNA, single and double stranded DNA and RNA, and antisense oligonucleotides into human and animal cells. These chimeric VSV-G protein-nucleic acid transfer vehicles have widespread applications to deliver nucleic acids for exon skipping and gene delivery for gene replacement in human and animals.

Claims

1. A chimeric protein comprising a vesicular stomatitis virus G glycoprotein (VSV-G) and a nucleic acid binding protein selected from the group consisting of histones, Single Strand DNA-Binding Protein-1 (SSBP-1), and RNaseIII.

2. The chimeric protein according to claim 1, wherein the nucleic acid binding protein is a histone.

3. The chimeric protein according to claim 2, wherein the histone is selected from the group consisting of: H2A, H2B, H3, and H4.

4. The chimeric protein according to claim 2, wherein the histone is tagged with VSV-G at the C-terminus.

5. The chimeric protein according to claim 2, wherein the histone is tagged with VSV-G at the N-terminus.

6. The chimeric protein according to claim 4, wherein the chimeric protein is one selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

7. The chimeric protein according to claim 5, wherein the chimeric protein is one selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22.

8. The chimeric protein according to claim 4, wherein the chimeric protein includes a sequence having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.

9. The chimeric protein according to claim 5, wherein the chimeric protein includes a sequence having at least 90% sequence identity with SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 22.

10. A therapeutic compound comprising the chimeric protein according to claim 1.

11. The chimeric protein according to claim 6, wherein the chimeric protein is encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5 and 7.

12. The chimeric protein according to claim 7, wherein the chimeric protein is encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 16, 18, 20 and 22.

13. The chimeric protein according to claim 8, wherein the chimeric protein is encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5 and 7.

14. The chimeric protein according to claim 9, wherein the chimeric protein is encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 16, 18, 20 and 22.

15. A chimeric protein consisting of a vesicular stomatitis virus G glycoprotein (VSV-G) and a nucleic acid binding protein selected from the group consisting of histones, SSBP-1 and RNase III.

16. A method of treating a medical condition in a subject comprising the steps of: providing a therapeutic compound comprising a chimeric protein comprising a vesicular stomatitis virus G glycoprotein (VSV-G) and a nucleic acid binding protein selected from the group consisting of histones, SSBP-1 and RNaseIII, and at least one nucleic acid; and administering to said subject a pharmaceutically active amount of said therapeutic compound.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The subject matter that is regarded as the invention is particularly pointed out and distinctly claimed in the claims at the conclusion of the specification. The foregoing and other objects, features, and advantages of the invention will be apparent from the following detailed description taken in conjunction with the accompanying drawings.

(2) FIG. 1 portrays chimeric VSV-G H2A protein fractions purified by SDS-PAGE analysis.

(3) FIG. 2 portrays western blot analysis of the proteins in the purified fractions from SDS-PAGE analysis as seen in FIG. 1.

(4) FIG. 3 portrays expression of GFP:HEK 293 cells transfected with eGFPN1 plasmid.

(5) FIG. 4A portrays GFP-including plasmid eGFPN1 transfected in HEK293 cells using purified VSV-G-H2A protein.

(6) FIG. 4B portrays GFP-including plasmid eGFPN1 transfected in NIH 3T3 cells using purified VSV-G-H2A protein.

(7) FIG. 5 portrays a method of treating a medical condition using a chimeric protein such as that isolated in FIG. 1.

DETAILED DESCRIPTION

(8) In some embodiments, the present disclosure is directed to a number of chimeric VSV-G (or VSV-G variants) proteins comprising VSV-G and at least one nucleic acid binding protein. In some embodiments, these proteins are used as transfer vehicles to enhance delivery of nucleic acids like plasmid DNA, single and double stranded DNA and RNA, and antisense oligonucleotides into human and animal cells.

(9) VSV-G cloned in expression plasmids, when transfected in cells, form sedimetable vesicles in the absence of any viral components. The chimeric proteins described here efficiently complex with nucleic acids in cell free systems and can be used as an effective means for delivering AOs and genes of interest in human and animal cells. This approach mitigates a number of risks and issues that are associated with gene therapy and exon skipping, i.e. there is no risk of toxicity related to viral production or risk of viral genome incorporation and possible mutations arising as a result. Since the VSV-G proteins enter into cells via the LDL receptors which are almost ubiquitously expressed, the transduction efficiency of the chimeric VSV-G-nucleic acid transfer vehicle is higher than that achieved by exon-skipping. The chimeric VSV-G-nucleic acid transfer vehicle consistent with some embodiments of the present disclosure can also replace the current mechanism of gene therapy. As this proposed chimeric VSV-G-nucleic acid transfer vehicle does not rely on virus production, it has fewer side effects and can be administered subcutaneously. This system can be used for gene replacement and can have wide application to cure many disorders arising from genetic mutations.

(10) In some embodiments, wild-type VSV-G is used in the chimeric protein. In some embodiments, VSV-G variants are used in the chimeric protein. In some embodiments, the VSV-G variants include the thermostable and serum resistant mutants of VSV-G, e.g. S162T, T230N, T368A, or combined mutants T230N+T368A or K66T+S162T+T230N+T368A. In some embodiments, variant VSV-G has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with wild-type VSV-G. As used in the following embodiments, the term “VSV-G” refers to both wild-type VSV-G and VSV-G variants.

(11) In some embodiments, the chimeric protein of the present disclosure has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the combined sequence of VSV-G+nucleic acid binding protein, with the nucleic acid binding protein tagged with VSV-G at the C-terminus. In some embodiments, chimeric protein has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the combined sequence of VSV-G+nucleic acid binding protein, with the nucleic acid binding protein tagged with VSV-G at the N-terminus. In some embodiments, the chimeric protein comprises a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with at least one of SEQ. ID NO.: 1, SEQ. ID NO.: 3, SEQ. ID NO.: 5, SEQ. ID NO.: 7, SEQ. ID NO.: 9, SEQ. ID NO.: 11, SEQ. ID NO.: 13, SEQ. ID NO.: 15, SEQ. ID NO.: 17, SEQ. ID NO.: 19, SEQ. ID NO.: 21, SEQ. ID NO.: 23, or SEQ. ID NO.: 25. In some embodiments, the chimeric protein comprises an amino acid sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with at least one of SEQ. ID NO.: 2, SEQ. ID NO.: 4, SEQ. ID NO.: 6, SEQ. ID NO.: 8, SEQ. ID NO.: 10, SEQ. ID NO.: 12, SEQ. ID NO.: 14, SEQ. ID NO.: 16, SEQ. ID NO.: 18, SEQ. ID NO.: 20, SEQ. ID NO.: 22, SEQ. ID NO.: 24, or SEQ. ID NO.: 26. In some embodiments, any suitable mutations, substitutions, additions, and deletions may be made to the chimeric protein so long as the pharmacological activity of the resulting variant chimeric protein is retained.

(12) In some embodiments, the nucleic acid binding protein is selected from the group consisting of H2A histone, H2B histone, H3 histone, H4 histone, SSBP-1, RNase III, and combinations thereof.

(13) SEQ. ID NO: 1 is a nucleotide sequence of an H2A histone-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(14) SEQ. ID NO: 2 is an amino acid sequence of an H2A histone-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(15) SEQ. ID NO: 3 is a nucleotide sequence of an H2B histone-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(16) SEQ. ID NO: 4 is an amino acid sequence of an H2B histone-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(17) SEQ. ID NO: 5 is a nucleotide sequence of an H3 histone-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(18) SEQ. ID NO: 6 is an amino acid sequence of an H3 histone-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(19) SEQ. ID NO: 7 is a nucleotide sequence of an H4 histone-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(20) SEQ. ID NO: 8 is an amino acid sequence of an H4 histone-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(21) SEQ. ID NO: 9 is a nucleotide sequence of an SSBP-1-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(22) SEQ. ID NO: 10 is an amino acid sequence of an SSBP-1-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(23) SEQ. ID NO: 11 is a nucleotide sequence of an RNase III-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(24) SEQ. ID NO: 12 is an amino acid sequence of an RNase III-VSV-G chimeric protein, with VSV-G at the C-terminus, consistent with some embodiments of the present disclosure.

(25) SEQ. ID NO: 13 is a nucleotide sequence of a partial RNase III-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(26) SEQ. ID NO: 14 is an amino acid sequence of a partial RNase III-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(27) SEQ. ID NO: 15 is a nucleotide sequence of an H2A histone-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(28) SEQ. ID NO: 16 is an amino acid sequence of an H2A histone-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(29) SEQ. ID NO: 17 is a nucleotide sequence of an H2B histone-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(30) SEQ. ID NO: 18 is an amino acid sequence of an H2B histone-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(31) SEQ. ID NO: 19 is a nucleotide sequence of an H3 histone-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(32) SEQ. ID NO: 20 is an amino acid sequence of an H3 histone-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(33) SEQ. ID NO: 21 is a nucleotide sequence of an H4 histone-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(34) SEQ. ID NO: 22 is an amino acid sequence of an H4 histone-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(35) SEQ. ID NO: 23 is a nucleotide sequence of an SSBP-1-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(36) SEQ. ID NO: 24 is an amino acid sequence of an SSBP-1-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(37) SEQ. ID NO: 25 is a nucleotide sequence of an RNase III-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(38) SEQ. ID NO: 26 is an amino acid sequence of an RNase III-VSV-G chimeric protein, with VSV-G at the N-terminus, consistent with some embodiments of the present disclosure.

(39) In some embodiments, the present disclosure is directed to a therapeutic compound comprising a chimeric protein consistent with those described in the above-identified embodiments. In some embodiments, as shown in FIG. 5, the present disclosure is directed to a method of treating a medical condition within a subject. In some embodiments, the method of treating a subject comprises the steps of providing 500 a therapeutic compound comprising a chimeric protein including VSV-G, a nucleic acid binding protein, and at least one nucleic acid, and administering 510 to the subject a pharmaceutically active amount of the therapeutic compound. In some embodiments, at least one nucleic acid is a therapeutic gene.

EXAMPLE

(40) The following example utilizes a VSV-G-H2A chimeric protein constructed from a human histone H2A protein tagged with VSV-G at the N-terminus. The VSV-G-H2A chimeric gene was synthesized using the propriety technology from Integrated DNA Technologies, Skokie, Ill. The VSV-G-H2A gene was cloned in the mammalian expression vector pTT5 at EcoRI and NotI restriction enzyme sites. The plasmid was prepared and sequenced for confirmation.

(41) HEK293T cells were passed to ˜70% confluency a day prior to transfection (3×T75 flasks, ˜7.5×10.sup.6 cells/flask). The following day, the cells in T75 flasks were transfected using Lipofectamine® 2000 (Life Technologies Corp., Carlsbad, Calif.) (per T75 flask: 3:1 ratio; 20 ug DNA; and 60 μL Lipofectamine® 2000). Flasks were incubated at 37° C. and 5% CO.sub.2 overnight. 24 hours after transfection, the conditioned media was removed and replaced with fresh media (14 mL/flask). Cells were further incubated overnight. Conditioned media was harvested and replaced with fresh media (14 mL/flask) and again incubated overnight. Harvested media was then filtered using 0.45 μm filter and stored at −80° C. The following day, conditioned media was harvested again and filtered using 0.45 μm filter. Conditioned media was pooled with media from the previous day (˜84 mL).

(42) Conditioned media was centrifuged using the Optima® Ultra Centrifuge (with swinging bucket rotor SW32Ti) (Beckman Coulter, Inc., Brea, Calif.) at 25,000 rpm for 2 h at 4° C. (3 centrifuge tubes, ˜28 mL/tube). Supernatant was removed and pellets were resuspended in 5 mL PBS per tube. 5 mL of 20% sucrose/PBS cushion plus 5 mL resuspended pellet was added to a new centrifuge tube. PBS was overlaid to fill the centrifuge tube. Samples were centrifuged at 25,000 rpm for 6 hours at 4° C. Supernatant was removed and each pellet was resuspended in 100 μL PBS (300 μL total volume). An additional 100 μL of PBS was added to each centrifuge tube to resuspend any remaining VSV-G-H2A protein (300 μL total volume). Protein concentration was measured by A660 Assay.

(43) The chimeric VSV-G H2A protein fractions thus purified were run on polyacrylamide gels before transfer to nitrocellulose membranes. Proteins were run in 4-15% BioRad TGX™ gel (BioRad Laboratories Inc., Hercules, Calif.) with BioRad Precision Plus Protein™ markers, at 300 V for 21 minutes and then stained with SYPRO®-Orange stain (Molecular Probes, Inc., Eugene, Oreg.), the results of which can be seen at FIG. 1. The contents for each lane found in FIG. 1 are as follows: Lane 1: Negative Control—untransfected cells only; Lane 2: molecular weight marker; Lane 3: M20336-01 (20 μL load); Lane 4: M20336-01 (2 μL load); Lane 5: molecular weight marker; and Lane 6: M20336-02 (20 μL load). The HEK293 untransfected lane did not stain for any protein while rest of the lanes containing the fractions of purified VSV-G-H2A chimeric protein stained for proteins confirming the presence of purified proteins in the fractions.

(44) After confirming the presence of the proteins in the purified fractions, proteins were run using the same conditions as described above and transferred to nitrocellulose membrane. The chimeric VSV-G-H2A protein was detected by probing with anti-VSV-G-primary antibody and anti-rabbit HRP secondary antibody. Proteins were transferred to nitrocellulose membrane using Bio-Rad Trans-Blot® Turbo™. Signal was detected using the SNAP id® system (Merck KGAA, Darmstadt, Del.) and SuperSignal® West Pico chemiluminescent substrate (Pierce Biotechnology, Inc., Rockford, Ill.), the results of which can be seen in the western blot shown in FIG. 2. The contents for each lane found in FIG. 2 are as follows: Lane 1: molecular weight marker; Lane 2: M20336-01 (20 μL load); Lane 3: molecular weight marker; Lane 4: M20336-01 (2 μL load); Lane 5: molecular weight marker; Lane 6: M20336-02 (20 μL load); Lane 7: molecular weight marker; Lane 8: Negative Control—untransfected cells only. A band was detected specific to the size of VSV-G H2A chimeric protein at 75 kD in lanes 2 and 6 containing 20 μL load of protein. No bands were detected in lanes 4 and 8 with 2 μL load of purified protein fraction and non-transfected HEK293 protein fraction. Therefore, the presence of VSV-G-H2A chimeric protein in the purified fraction was confirmed.

(45) In order to evaluate the capacity of the purified VSV-G-H2A chimeric protein to act as nucleic acid transfer vehicle, HEK293 cells and NIH 3T3 cells were transfected with green fluorescent protein (GFP) expressing plasmid eGFPN1 utilizing the VSV-G-H2A chimeric protein. Firstly, the eGFPN1 plasmid was transfected in HEK293 cells using ViaFect™ transfection reagent (Promega Corp., Madison, Wis.) to confirm that GFP was expressed properly. Successful GFP expression is shown in FIG. 3.

(46) To determine whether similar expression of GFP could be seen when VSV-G-H2A chimeric protein was used as a transfer vehicle, 2 μg of eGFPN1 plasmid was mixed with 3 μg of VSV-G H2A purified chimeric protein and overlaid in each of HEK293 and NIH 3T3 cells seeded on coverslips in 6-well plates. Cells were incubated for 48 hours before analysis. To detect whether GFP has expressed, the existing medium in the cells was aspirated, washed in Dulbecco's phosphate buffered saline (DPBS), and then fixed in 4% paraformaldehyde solution. Cells were washed again with DPBS a couple of times, stained with 4′,6-diamidino-2-phenylindole (DAPI), and then mounted in appropriate mounting medium and viewed under a fluorescence microscope. The results of this procedure can be seen in FIGS. 4A and 4B, wherein DAPI staining depicts the nucleus and the green fluorescence depicts the GFP. Interestingly, the HEK293 and NIH 3T3 cells in which VSV-G-H2A purified chimeric protein was used as a transfer vehicle to transfect eGFPN1 plasmid expressed GFP. Therefore, it was concluded that VSV-G-H2A chimeric protein, as well as the other chimeric proteins disclosed in the present disclosure and functional equivalents thereof, are candidates for use as nucleic acid transfer vehicles as proposed by the present disclosure.

(47) One or more embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.