1111111111111111 IIIIII IIIII 1111111111 11111 11111 111111111111111 IIIII IIIII IIIIII IIII 11111111
`US 20180036429Al
`
`c19) United States
`c12) Patent Application Publication
`Acharjee
`
`c10) Pub. No.: US 2018/0036429 Al
`Feb. 8, 2018
`(43) Pub. Date:
`
`(54) CHIMERIC VSV-G PROTEINS AS NUCLEIC
`ACID TRANSFER VEHICLES
`
`(71) Applicant: Serendipity Biotech Inc., New York,
`NY (US)
`
`(72)
`
`Inventor: Sujata Acharjee, New York, NY (US)
`
`(73) Assignee: Serendipity Biotech Inc., New York,
`NY (US)
`
`(21) Appl. No.: 15/791,262
`
`(22)
`
`Filed:
`
`Oct. 23, 2017
`
`Related U.S. Application Data
`
`(63)
`
`(60)
`
`Continuation of application No. 14/695,265, filed on
`Apr. 24, 2015, now Pat. No. 9,821,076.
`
`Provisional application No. 61/984,290, filed on Apr.
`25, 2014.
`
`Publication Classification
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(51)
`
`(52)
`
`Int. Cl.
`A61K 48100
`C07K 141005
`C07K 141435
`C12N 9122
`C07K 14147
`U.S. Cl.
`CPC ...... A61K 4810008 (2013.01); C07K 2319/85
`(2013.01); C07K 2319/80 (2013.01); C07K
`2319/01 (2013.01); C12N 9122 (2013.01);
`C07K 1414722 (2013.01); C07K 141435
`(2013.01); C07K 141005 (2013.01)
`ABSTRACT
`(57)
`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 oligo(cid:173)
`nucleotides into human and animal cells. These chimeric
`VSV-G protein-nucleic acid transfer vehicles have wide(cid:173)
`spread applications to deliver nucleic acids for exon skip(cid:173)
`ping and gene delivery for gene replacement in human and
`animals.
`
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`providing a therapeutic compound comprising a chimeric protein
`including VSV-G, a nucleic acid binding protein, and at least one ~ 500
`nucleic acid
`
`administering to the subject a pharmaceutically active amount of
`the therapeutic compound
`
`510
`~
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`FIG. 5
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`CHIMERIC VSV-G PROTEINS AS NUCLEIC
`ACID TRANSFER VEHICLES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application claims the benefit of U.S. Provi(cid:173)
`sional Patent No. 61/984,290, filed Apr. 25, 2014, which is
`incorporated by reference herein in its entirety.
`
`FIELD OF THE DISCLOSURE
`
`[0002] What is disclosed is a chimeric or fusion protein
`including a membrane transport domain and a nucleic acid
`binding domain allowing targeted delivery of nucleic acids
`in humans and animals for the treatment of medical condi(cid:173)
`tions.
`
`BACKGROUND OF THE DISCLOSURE
`
`[0003] The vesicular stomatitis virus G glycoprotein
`(hereinafter referred to as "VSV-G") is widely used to
`pseudotype viral vectors due to its wide tropism and stabil(cid:173)
`ity. These viral vectors facilitate gene transduction in human
`and animals. The VSV-G proteins, when not associated with
`any viral vectors, are also alone capable of forming com(cid:173)
`plexes with naked plasmid DNA in cell free conditions
`which can be transfected to cells thereafter.
`[0004] The fusogenic G glycoprotein of the vesicular
`stomatitis virus has proved to be a useful tool for viral(cid:173)
`mediated gene delivery by acting as an envelope protein.
`Due to its wide tropism, VSV-G has been used as an efficient
`surrogate envelope protein to produce more stable and high
`titer pseudotyped murine leukemia virus (MLV)-based ret(cid:173)
`rovirus and lentivirus-based vectors, all of which have been
`effectively used for gene therapy. The reason behind this
`pantropism of VSV remained elusive for a long period.
`Recently, it has been found that the VSV enters the cell
`through a highly ubiquitous low-density lipoprotein (LDL)
`receptor having wide distribution.
`[0005] However, there are some limitations associated
`with the use of VSV-G. It is cytotoxic to producer cells,
`though the use of tetracycline-regulated promoters has
`helped to overcome this problem. In addition, serum inac(cid:173)
`tivation ofVSV-G pseudotyped viruses poses a problem and
`impedes their function to some extent in vivo. To overcome
`the latter problem, VSV-G mutants have been generated
`which are more thermostable as well as serum-resistant.
`VSV-G mutants harboring T230N+T368A or K66T+
`S 162T + T230N + T368A mutations exhibited more resistance
`to serum inactivation and higher thermostability.
`[0006] Apart from acting as a fusogenic envelope protein
`for many viral vectors, previous studies showed that purified
`soluble VSV-G itself can be inserted into lipid bilayers of
`liposomes and lipid vesicles in cell free system in vitro.
`Additionally, it has been shown that VSV-G can form a
`complex with naked plasmid DNA in the absence of any
`transfection reagent and can thereby enhance the transfec(cid:173)
`tion of naked plasmid DNA into cells. Sucrose gradient
`sedimentation analysis demonstrated that VSV-G associates
`with plasmid DNA and MLV gag-pol particles to form
`ternary complexes that co-sediment with high DNA trans(cid:173)
`fecting activity. This transfection could be abolished by
`adding antibody for VSV-G.
`In eukaryotic cells, heritable genetic material is
`[0007]
`packaged into structures known as chromatin consisting of
`
`DNA and protein. The basic repeating unit of chromatin is
`the nucleosome core, which consists of 147 base pairs of
`DNA wrapped in 1.7 left-handed superhelical turns around
`the surface of an octameric protein core formed by two
`molecules each of histones H2A, H2B, H3, and H4. His(cid:173)
`tones are highly basic proteins that bind very avidly and
`non-specifically to nucleic acids. Histones were among the
`first proteins studied due to their relative ease of isolation
`and all four histone proteins (H2A, H2B, H3, and H4) can
`be expressed in bacteria. This has allowed purifying and
`reconstituting of the histone proteins in cell free systems
`using well defined protocols. Though the native histone
`proteins undergo an extensive array of posttranslational
`modifications, recombinant histones do not undergo post(cid:173)
`translational modifications and can be obtained in a highly
`pure form due to their high expression levels.
`[0008] Single Strand DNA-Binding Proteins (hereinafter
`referred to as "SSBP") are ubiquitously expressed and
`involved in a variety of DNA metabolic processes including
`replication, recombination, damage, and repair. SSBP-1 is a
`housekeeping gene involved in mitochondrial biogenesis. It
`is also a subunit of a single-stranded DNA (ssDNA)-binding
`complex involved in the maintenance of genome stability.
`[0009] Ribonuclease III (hereinafter referred to as "RNase
`III") is an enzyme that is expressed in most of the cells and
`is involved in the processing of pre-rRNA. It has a catalytic
`domain and an RNA binding domain that is located in the
`C-terminal end of the enzyme. Inhibition of human RNase
`III resulted in cell death suggesting a very important role of
`this enzyme.
`[0010] Gene therapy and exon skipping have served as a
`means of gene transduction or gene manipulation respec(cid:173)
`tively in humans during the past two decades. Gene therapy
`and exon skipping were initially developed as therapeutic
`strategies focused to address detrimental monogenetic dis(cid:173)
`eases for which there were no available options for treat(cid:173)
`ment, e.g. primary immunodeficiency. These approaches
`later found widespread application in curing neurodegen(cid:173)
`erative diseases, cancer, metabolic disorders, and more.
`[0011] Gene therapy involves delivery of genes of interest
`cloned in viral vectors which are capable of producing
`viruses when transduced in human cells. Despite the con(cid:173)
`tinuous improvement of retroviral and lentiviral gene trans(cid:173)
`fer systems for gene delivery during the last many years,
`there remain severe limitations preventing the development
`of efficient and safe clinical applications for these systems.
`These limitations include: their inability to target infection
`to cells of interest, inefficient transduction, propensity of
`viral vectors to get incorporated in human genome and
`create mutations, elicited high immune responses, inability
`to be administered intravenously or subcutaneously, and
`intramuscular administration that only leads to local delivery
`of the gene. Owing to these limitations, no gene therapy
`based medication has been approved by FDA for use in
`humans, though there have been many clinical trials during
`the past two decades and also many ongoing clinical trials.
`[0012] Exon skipping is a therapeutic strategy where anti(cid:173)
`sense oligonucleotides (AO) are delivered in humans to
`modulate splicing of genes resulting in mRNA that either
`produces functional proteins or blocks their production. A Os
`are short nucleic acid sequences designed to selectively bind
`to specific mRNA or pre-mRNA sequences. Despite the very
`convincing underlying principle behind this strategy, only
`one AO has been approved by the FDA (Vitravene™, an
`
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`intraocular injection to inhibit cytomegalovirus retinitis in
`immunocompromised patients; Isis Pharmaceuticals, Carls(cid:173)
`bad, Calif.), and this drug is no longer marketed. There are
`certain limitations associated with the use of AOs including
`difficulty in achieving pharmacologically significant con(cid:173)
`centrations in cells due to biological barriers like endothelial
`and basement membrane, cell membrane, and sequestration
`by phagolysosomes.
`[0013] Further discussion on the subjects of gene transfer
`and delivery may be found in U.S. Pat. No. 7,531,647
`("Lentiviral Vectors for Site-Specific Gene Insertion"); U.S.
`Pat. No. 8,158,827 ("Transfection Reagents"); and U.S. Pat.
`No. 8,652,460 ("Gene Delivery System and Method of
`Use") and U.S. patent application Ser. No. 14/635,012
`("Chimeric Dystrophin-VSV-G Protein to Treat Dystrophi(cid:173)
`nopathies". The disclosures of each of U.S. Pat. Nos. 7,531,
`647, 8,158,827 and 8,652,460 and U.S. application Ser. No.
`14/635,012 are incorporated by reference herein in their
`entireties.
`[0014] The details of one or more embodiments of the
`invention are set forth in the accompanying drawings and
`the description below. Other features, objects and advan(cid:173)
`tages of the invention will be apparent from the description
`and drawings, and from the claims.
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0015] Disclosed herein is a chimeric protein incorporat(cid:173)
`ing a transport domain and a nucleic acid binding domain
`and methods of utilizing those chimeric proteins for targeted
`delivery of therapeutic nucleic acids.
`[0016]
`In some embodiments, the present disclosure is
`directed to a chimeric protein comprising VSV-G and a
`nucleic acid binding protein. In some embodiments, the
`nucleic acid binding protein is a histone. In some embodi(cid:173)
`ments, the histone is selected from the group consisting of:
`H2A, H2B, H3, and H4. In some embodiments, the histone
`is tagged with VSV-G at the C-terminus. In some embodi(cid:173)
`ments, histone is tagged with VSV-G at the N-terminus.
`[0017]
`In some embodiments, the chimeric protein com(cid:173)
`prises SEQ. ID NO.: 1, SEQ. ID NO.: 2, SEQ. ID NO.: 3,
`SEQ. ID NO.: 4, SEQ. ID NO.: 5, SEQ. ID NO.: 6, SEQ. ID
`NO.: 7, or SEQ. ID NO.: 8, and pharmacologically accept(cid:173)
`able equivalents thereof. In some embodiments, the chimeric
`protein comprises SEQ. ID NO.: 15, SEQ. ID NO.: 16, SEQ.
`ID NO.: 17, SEQ. ID NO.: 18, SEQ. ID NO.: 19, SEQ. ID
`NO.: 20, SEQ. ID NO.: 21, or SEQ. ID NO.: 22, and
`pharmacologically acceptable equivalents thereof. In some
`embodiments, the chimeric protein includes a sequence
`having at least 90%, at least 95%, at least 96%, at least 97%,
`at least 98%, or at least 99% sequence identity with SEQ. ID
`NO.: 1, SEQ. ID NO.: 2, SEQ. ID NO.: 3, SEQ. ID NO.: 4,
`SEQ. ID NO.: 5, SEQ. ID NO.: 6, SEQ. ID NO.: 7, or SEQ.
`ID NO.: 8. In some embodiments, the chimeric protein
`includes a sequence having at least 90%, at least 95%, at
`least 96%, at least 97%, at least 98%, or at least 99%
`sequence identity with SEQ. ID NO.: 15, SEQ. ID NO.: 16,
`SEQ. ID NO.: 17, SEQ. ID NO.: 18, SEQ. ID NO.: 19, SEQ.
`ID NO.: 20, SEQ. ID NO.: 21, or SEQ. ID NO.: 22.
`[0018]
`In some embodiments, the nucleic acid binding
`protein is SSBP-1. In some embodiments, SSBP-1 is tagged
`with VSV-G at the C-terminus. In some embodiments,
`SSBP-1 is tagged with VSV-G at the N-terminus. In some
`embodiments, the chimeric protein comprises SEQ. ID NO.:
`9 or SEQ. ID NO.: 10, and pharmacologically acceptable
`
`equivalents thereof. In some embodiments, the chimeric
`protein comprises SEQ. ID NO.: 23 or SEQ. ID NO.: 24, and
`pharmacologically acceptable equivalents thereof. In some
`embodiments, the chimeric protein includes a sequence
`having at least 90%, at least 95%, at least 96%, at least 97%,
`at least 98%, or at least 99% sequence identity with SEQ. ID
`NO.: 9 or SEQ. ID NO.: 10. In some embodiments, the
`chimeric protein includes a sequence having at least 90%, at
`least 95%, at least 96%, at least 97%, at least 98%, or at least
`99% sequence identity with SEQ. ID NO.: 23 or SEQ. ID
`NO.: 24.
`[0019]
`In some embodiments, the nucleic acid binding
`protein is RNase III. In some embodiments, RNase III is
`tagged with VSV-G at the C-terminus. In some embodi(cid:173)
`ments, RNase III is tagged with VSV-G at the N-terminus.
`In some embodiments, the chimeric protein comprises SEQ.
`ID NO.: 11, SEQ. ID NO.: 12, or SEQ. ID NO.: 13, and
`pharmacologically acceptable equivalents thereof. In some
`embodiments, wherein the chimeric protein comprises SEQ.
`ID NO.: 14, SEQ. ID NO.: 25, or SEQ. ID NO.: 26, and
`pharmacologically acceptable equivalents thereof. In some
`embodiments, wherein the chimeric protein includes a
`sequence having at least 90%, at least 95%, at least 96%, at
`least 97%, at least 98%, or at least 99% sequence identity
`with SEQ. ID NO.: 11, SEQ. ID NO.: 12, or SEQ. ID NO.:
`13. In some embodiments, wherein the chimeric protein
`includes a sequence having at least 90%, at least 95%, at
`least 96%, at least 97%, at least 98%, or at least 99%
`sequence identity with SEQ. ID NO.: 14, SEQ. ID NO.: 25,
`or SEQ. ID NO.: 26.
`[0020]
`In some embodiments, the present disclosure is
`directed to a method of treating a medical condition in a
`subject comprising the steps of providing a therapeutic
`compound comprising a chimeric protein including VSV-G,
`a nucleic acid binding protein, and at least one nucleic acid,
`and administering to said subject a pharmaceutically active
`amount of said therapeutic compound. In some embodi(cid:173)
`ments, the present disclosure is directed to a therapeutic
`compound comprising a chimeric protein as described
`herein.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0021] 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.
`[0022] FIG. 1 portrays chimeric VSV-G H2A protein
`fractions purified by SDS-PAGE analysis.
`[0023] FIG. 2 portrays western blot analysis of the pro(cid:173)
`teins in the purified fractions from SDS-PAGE analysis as
`seen in FIG. 1.
`[0024] FIG. 3 portrays expression of GFP:HEK 293 cells
`transfected with eGFPNl plasmid.
`[0025] FIG. 4A portrays GFP-including plasmid eGFPNl
`transfected in HEK293 cells using purified VSV-G-H2A
`protein.
`[0026] FIG. 4B portrays GFP-including plasmid eGFPNl
`transfected in NIH 3T3 cells using purified VSV-G-H2A
`protein.
`[0027] FIG. 5 portrays a method of treating a medical
`condition using a chimeric protein such as that isolated in
`FIG. 1.
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`DETAILED DESCRIPTION
`
`In some embodiments, the present disclosure is
`[0028]
`directed to a number of chimeric VSV-G (or VSV-G vari(cid:173)
`ants) 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.
`[0029] VSV-G cloned in expression plasmids, when trans(cid:173)
`fected 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(cid:173)
`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.
`In some embodiments, wild-type VSV-G is used in
`[0030]
`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 resis(cid:173)
`tant 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.
`In some embodiments, the chimeric protein of the
`[0031]
`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 embodi(cid:173)
`ments, 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. IDNO.: 16, SEQ. IDNO.: 18, SEQ. IDNO.: 20, SEQ.
`ID NO.: 22, SEQ. ID NO.: 24, or SEQ. ID NO.: 26. In some
`embodiments, any suitable mutations, substitutions, addi(cid:173)
`tions, and deletions may be made to the chimeric protein so
`long as the pharmacological activity of the resulting variant
`chimeric protein is retained.
`In some embodiments, the nucleic acid binding
`[0032]
`protein is selected from the group consisting ofH2Ahistone,
`H2B histone, H3 histone, H4 histone, SSBP-1, RNase III,
`and combinations thereof.
`[0033] SEQ. ID NO: 1 is a nucleotide sequence of an H2A
`histone-VSV-G chimeric protein, with VSV-G at the C-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0034] 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 pres(cid:173)
`ent disclosure.
`[0035] SEQ. ID NO: 3 is a nucleotide sequence of an H2B
`histone-VSV-G chimeric protein, with VSV-G at the C-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0036] 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 pres(cid:173)
`ent disclosure.
`[0037] SEQ. ID NO: 5 is a nucleotide sequence of an H3
`histone-VSV-G chimeric protein, with VSV-G at the C-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0038] SEQ. ID NO: 6 is an amino acid sequence of an H3
`histone-VSV-G chimeric protein, with VSV-G at the C-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0039] SEQ. ID NO: 7 is a nucleotide sequence of an H4
`histone-VSV-G chimeric protein, with VSV-G at the C-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0040] SEQ. ID NO: 8 is an amino acid sequence of an H4
`histone-VSV-G chimeric protein, with VSV-G at the C-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0041] SEQ. ID NO: 9 is a nucleotide sequence of an
`SSBP-1-VSV-G chimeric protein, with VSV-G at the C-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0042] SEQ. ID NO: 10 is an amino acid sequence of an
`SSBP-1-VSV-G chimeric protein, with VSV-G at the C-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0043] 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 pres(cid:173)
`ent disclosure.
`[0044] 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 pres(cid:173)
`ent disclosure.
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`[0045] 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.
`[0046] 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.
`[0047] 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 pres(cid:173)
`ent disclosure.
`[0048] 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 pres(cid:173)
`ent disclosure.
`[0049] 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 pres(cid:173)
`ent disclosure.
`[0050] 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 pres(cid:173)
`ent disclosure.
`[0051] SEQ. ID NO: 19 is a nucleotide sequence ofan H3
`histone-VSV-G chimeric protein, with VSV-G at the N-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0052] 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 pres(cid:173)
`ent disclosure.
`[0053] SEQ. ID NO: 21 is a nucleotide sequence of an H4
`histone-VSV-G chimeric protein, with VSV-G at the N-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0054] 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 pres(cid:173)
`ent disclosure.
`[0055] SEQ. ID NO: 23 is a nucleotide sequence of an
`SSBP-1-VSV-G chimeric protein, with VSV-G at the N-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0056] SEQ. ID NO: 24 is an amino acid sequence of an
`SSBP-1-VSV-G chimeric protein, with VSV-G at the N-ter(cid:173)
`minus, consistent with some embodiments of the present
`disclosure.
`[0057] 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 pres(cid:173)
`ent disclosure.
`[0058] 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 pres(cid:173)
`ent disclosure.
`[0059]
`In some embodiments, the present disclosure is
`directed to a therapeutic compound comprising a chimeric
`protein consistent with those described in the above-identi(cid:173)
`fied 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 chime-
`
`ric 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
`
`[0060] 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(cid:173)
`H2A chimeric gene was synthesized using the propriety
`technology from Integrated DNA Technologies, Skokie, IL.
`The VSV-G-H2A gene was cloned in the mammalian
`expression vector pTT5 at EcoRI and Natl restriction
`enzyme sites. The plasmid was prepared and sequenced for
`confirmation.
`[0061] HEK293 T cells were passed to - 70% confluency a
`day prior to transfection (3x T75 flasks, -7.5xl0 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 incu(cid:173)
`bated at 37° C. and 5% CO2 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 previ(cid:173)
`ous day (-84 mL).
`[0062] 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 remain(cid:173)
`ing VSV-G-H2A protein (300 µL total volume). Protein
`concentration was measured by A660 Assay.
`[0063] 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% Bio(cid:173)
`Rad 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-untrans(cid:173)
`fected 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.
`
`Page 11 of 61
`
`

`

`US 2018/0036429 Al
`
`Feb. 8,2018
`
`5
`
`[0064] After confirming the presence of the proteins in the
`purified fractions, proteins were run using the same condi(cid:173)
`tions as described above and transferred to nitrocellulose
`membrane. The chimeric VSV-G-H2Aprotein was detected
`by probing with anti-VSV-G-primary antibody and anti(cid:173)
`rabbit HRP secondary antibody. Proteins were transferred to
`nitrocellulose membrane using Bio-Rad Trans-Blot® Tur(cid:173)
`bo™. 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 Con(cid:173)
`trol-untransfected cells only. A band was detected specific
`to the size ofVSV-G H2A chimeric protein at 7 5 kD in lanes
`2 and 6 containing 204 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 ofVSV-G-H2A chimeric protein in
`the purified fraction was confirmed.
`In order to evaluate the capacity of the purified
`[0065]
`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
`eGFPNl utilizing the VSV-G-H2A chimeric protein. Firstly,
`the eGFPNl plasmid was transfected in HEK293 cells using
`ViaFect™ transfection reagent (Promega Corp., Madison,
`
`SEQUENCE LISTING
`
`Wis.) to confirm that GFP was expressed properly. Success(cid:173)
`ful GFP expression is shown in FIG. 3.
`[0066] 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 eGFPNl 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 fluores(cid:173)
`cence 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. Inter(cid: