WO2011146885A2

WO2011146885A2 - Compositions and methods for lentiviral expression of apoa-1 or variants thereof using spliceosome mediated rna trans-splicing

Info

Publication number: WO2011146885A2
Application number: PCT/US2011/037419
Authority: WO
Inventors: Madaiah Puttaraju; Gary Stephen Mansfield; Nikolay Korokhov; Jenice G. D'costa; Laurent M. Humeau; Gerard J. Mcgarrity; Jun Wang
Original assignee: Virxsys Corporation
Priority date: 2010-05-21
Filing date: 2011-05-20
Publication date: 2011-11-24
Also published as: WO2011146885A3

Abstract

The disclosure provides methods and compositions for generating nucleic acid molecules through RNA trans-splicing that target a highly expressed pre-mRNA and contain the coding sequence of a protein or polypeptide of interest. The compositions disclosed include recombinant lentiviral vectors expressing pre-trans-splicing molecules (PTMs) designed to interact with the target precursor messenger RNA molecule (target premRNA) that is abundantly expressed, and mediate a trans-splicing reaction resulting in the generation of chimeric RNA molecule (chimeric RNA) capable of encoding a protein or polypeptide of interest.

Description

COMPOSITIONS AND METHODS FOR LENTIVIRAL EXPRESSION OF APOA-1 OR VARIANTS THEREOF USING SPLICEOSOME

MEDIATED RNA TRANS-SPLICING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional application Serial No.

61/347,349, filed 21 May 2010, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] This application relates to methods and compositions for generating novel nucleic acid molecules through RNA trans-splicing that target abundantly expressed precursor messenger RNA molecule (target pre-mRNA) and contain the coding sequence of a protein or polypeptide of interest. In particular, this application relates to methods and compositions for the expression of ApoA-1 by spliceosome mediated RNA irans-splicing, and, more particularly, to methods and compositions comprising lentiviral vectors containing pre-trans- splicing molecules (PTMs) to express ApoA- 1 via spliceosome mediated RNA irans-splicing (SMaRT™).

BACKGROUND OF THE INVENTION

RNA Splicing

[0003] DNA sequences in the chromosome are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns). Introns are removed from pre-mRNAs in a precise process called cis-splicing (Chow et al., 1977, Cell 12: 1-8; and Berget, S. M. et al., 1977, Proc. Natl. Acad. Sci. USA 74:3171-3175). Splicing takes place as a coordinated interaction of several small nuclear ribonucleoprotein particles (snRNP's) and many protein factors that assemble to form an enzymatic complex known as the spliceosome (Moore et al., 1993, in The RNA World, R. F. Gestland and J. F. Atkins eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Kramer, 1996, Annu. Rev. Biochem., 65:367-404; Staley and Guthrie, 1998, Cell 92:315-326).

[0004] In most cases, the splicing reaction occurs within the same pre-mRNA molecule, which is termed cis-splicing. Splicing between two independently transcribed pre-mRNAs is termed trans-splicing. Trans-splicing was first discovered in trypanosomes (Sutton & Boothroyd, 1986, Cell 47:527; Murphy et al., 1986, Cell 47:517) and subsequently in nematodes (Krause & Hirsh, 1987, Cell 49:753); flatworms (Rajkovic et al., 1990, Proc. Natl. Acad. Sci. USA, 87:8879; Davis et al., 1995, J. Biol. Chem. 270:21813) and in plant mitochondria (Malek et al., 1997, Proc. Natl. Acad. Sci. USA 94:553). In the parasite Trypanosoma brucei, all mRNAs acquire a splice leader (SL) RNA at their 5' termini by trans-splicing. A 5' leader sequence is also trans-spliced onto some genes in Caenorhabditis elegans. This mechanism is appropriate for adding a single common sequence to many different transcripts.

[0005] The mechanism of splice leader trans-splicing, which is nearly identical to that of conventional cis-splicing, proceeds via two phosphoryl transfer reactions. The first causes the formation of a 2'-5' phosphodiester bond producing a Ύ" shaped branched intermediate, equivalent to the lariat intermediate in cis-splicing. The second reaction, exon ligation, proceeds as in conventional cis-splicing. In addition, sequences at the 3' splice site and some of the snRNPs which catalyze the trans-splicing reaction, closely resemble their counterparts involved in cis-splicing.

[0006] Trans-splicing refers to a different process, where an intron of one pre-mRNA interacts with an intron of a second pre-mRNA, enhancing the recombination of splice sites between two conventional pre-mRNAs. This type of trans-splicing was postulated to account for transcripts encoding a human immunoglobulin variable region sequence linked to the endogenous constant region in a transgenic mouse (Shimizu et al., 1989, Proc. Natl. Acad. Sci. USA 86:8020). In addition, trans-splicing of c-myb pre-mRNA has been demonstrated (Vellard, M. et al. Proc. Natl. Acad. Sci., 1992 89:2511-2515) and RNA transcripts from cloned SV40 trans-spliced to each other were detected in cultured cells and nuclear extracts (Eul et al., 1995, EMBO. J 14:3226). However, naturally occurring trans-splicing of mammalian pre-mRNAs is thought to be a rare event (Flouriot G. et al., 2002 J. Biol. Chem: Finta, C. et al., 2002 J Biol Chem 277:5882-5890).

[0007] In vitro trans-splicing has been used as a model system to examine the mechanism of splicing by several groups (Konarska & Sharp, 1985, Cell 46: 165-171 Solnick, 1985, Cell 42:157; Chiara & Reed, 1995, Nature 375:510; Pasman and Garcia-Blanco, 1996, Nucleic Acids Res. 24:1638). Reasonably efficient trans-splicing (30% of cis-spliced analog) was achieved between RNAs capable of base pairing to each other, splicing of RNAs not tethered by base pairing was further diminished by a factor of 10. Other in vitro trans-splicing reactions not requiring obvious RNA-RNA interactions among the substrates were observed by Chiara & Reed (1995, Nature 375:510), Bruzik J. P. & Maniatis, T. (1992, Nature 360:692) and Bruzik J. P. and Maniatis, T., (1995, Proc. Natl. Acad. Sci. USA 92:7056- 7059). These reactions occur at relatively low frequencies and require specialized elements, such as a downstream 5' splice site or exonic splicing enhancers.

[0008] In addition to splicing mechanisms involving the binding of multiple proteins to the precursor mRNA which then act to correctly cut and join RNA, a third mechanism involves cutting and joining of the RNA by the intron itself, by what are termed catalytic RNA molecules or ribozymes. The cleavage activity of ribozymes has been targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. Upon hybridization to the target RNA, the catalytic region of the ribozyme cleaves the target. It has been suggested that such ribozyme activity would be useful for the inactivation or cleavage of target RNA in vivo, such as for the treatment of human diseases characterized by production of foreign of aberrant RNA. In such instances small RNA molecules are designed to hybridize to the target RNA and by binding to the target RNA prevent translation of the target RNA or cause destruction of the RNA through activation of nucleases. The use of antisense RNA has also been proposed as an alternative mechanism for targeting and destruction of specific RNAs.

[0009] Using the Tetrahymena group I ribozyme, targeted trans-splicing was

demonstrated in E. coli. (Sullenger B. A. and Cech. T. R., 1994, Nature 341:619-622), in mouse fibroblasts (Jones, J. T. et al., 1996, Nature Medicine 2:643-648), human fibroblasts (Phylacton, L. A. et al. Nature Genetics 18:378-381) and human erythroid precursors (Lan et al., 1998, Science 280:1593-1596). For a review of clinically relevant technologies to modify RNA see Sullenger and Gilboa, 2002 Nature 418:252-8. The present invention relates to the use of targeted trans-splicing mediated by native mammalian splicing machinery, i.e., spliceosomes, to reprogram or alter the coding sequence of a targeted mRNA.

[0010] U.S. Patent Nos. 6,083,702, 6,013,487 and 6,280,978 describe the use of PTMs to mediate a trans-splicing reaction by contacting a target precursor mRNA to generate novel chimeric mRNAs.

Cardiovascular Disease

[0011] Cardiovascular disease (CVD) is the most common cause of death in the Western societies, and its prevalence is increasing worldwide. One of the strongest predictors of risk is the plasma concentration of high-density lipoprotein (HDL) or apolipoprotein Al (apoA-1), the major protein component of HDL, which exhibits an inverse relationship with the development of atherosclerosis and coronary heart disease (Sirtori C R et al., 1999,

Atherosclerosis 142:29-40; Genest J 2003, J Inherit. Metab. Dis. 26:267-287). ApoA-1 is the major apolipoprotein of HDL and is a relatively abundant plasma protein with a

concentration of 1.0-1.5 mg/ml. ApoA-1 plays an important role in promoting the efflux of excess cholesterol from peripheral cells and tissues for transfer to the liver for excretion, a process called reverse cholesterol transport (RCT). Numerous in vitro and in vivo studies have demonstrated the protective effects of apoA-1 and HDL against atherosclerosis plaque development (Rubin E M, et al., Nature. 1991, 353:265-7; Plump A S et al., 1994 Proc Natl Acad. Sci. USA 91:9607-11; Paszty C, et al., 1994 J Clin Invest. 94:899-903; Duverger N et al., 1996, Circulation 94:713-7).

[0012] ApoA-1 Milano is one of a number of naturally occurring variants of wild type apoA-1. It was first identified in 1980 in an Italian family (Franceschini G et al., 1980, J. Clin. Invest. 66:892-900; Weisgraber K H et al., 1980 J Clin Invest. 66:901-907). To date 40 carriers have been identified and all are heterozygous. These carriers have low plasma HDL- cholesterol levels and moderately elevated levels of triglycerides, a condition that is usually associated with high-risk predictors for coronary heart disease. Despite severe reductions in plasma HDL-cholesterol levels and apoA-1 concentrations, the affected carriers do not develop coronary artery disease. In fact, infusions of the purified recombinant apoA- 1 Milano or expression of apoA- 1 Milano in rabbits and apoE deficient mice show protection against plaque formation and atherosclerosis (Ameli S et al., 1994, Circulation 90: 1935-41; Soma M R et al., 1995 Cir. Res. 76:405-11; Shah P K et al., 1998 Circulation 97:780-5; Franceschini G et al., 1999, Arterioscler Thromb Vase Biol. 19: 1257-1262; Chiesa G et al., 2002, Cir. Res. 90:974-80; Chiesa G and Sirtori C, 2003, Curr. Opin. Lipdol. 14: 159-163). Results from clinical trials, however have shown more modest levels of reduction. The degree of plaque reduction may be related to the limited number of doses and amounts of protein administered, and/or its duration in the circulation (pharmacokinetics).

[0013] Plasma apoA-1 is a single polypeptide chain of 243 amino acids, whose primary sequence is known (Brewer et al, 1978, Biochem. Biophys. Res. Commun. 80:623-630). ApoA-1 is synthesized as a 267 amino acid precursor in the cell. This preproapolipoproteinA- 1 is first intracellularly processed by N-terminal cleavage of 18 amino acids to yield proapolipoproteinA-1, and then further cleavage of 6 amino acids in the plasma or the lymph by the activity of specific proteases to yield mature apolipoproteinA-1. The major structural requirement of the apoA-1 molecule is believed to be the presence of repeat units of 11 or 22 amino acids, presumed to exist in amphipathic helical conformation (Segrest et al., 1974, FEBS Lett 38:247-253). This structure allows for the main biological activities of apoA-1, i.e. lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation.

[0014] Human apolipoproteinAl Milano (apoA-1 Milano) is a natural variant of ApoA-1 (Weisgraber et al, 1980, J. Clin. Invest 66:901-907). In apoA-1 Milano the amino acid Argl73 is replaced by the amino acid Cysl73. Since apoA-1 Milano contains one Cys residue per polypeptide chain, it may exist in a monomeric, homodimeric, or heterodimeric form. These forms are chemically interchangeable, and the term apoA- 1 Milano does not, in the present context, discriminate between these forms. On the DNA level the variant form results from a C to T substitution in the gene sequence, i.e. the codon CGC changed to TGC, allowing the translation of a Cys instead of Arg at amino acid position 173. However, this variant of apoA-1 is one of the most interesting variants, in that apoA-1 Milano subjects are characterized by a remarkable reduction in HDL-cholesterol level, but without an apparent increased risk of arterial disease (Franceschini et al. 1980, J. Clin. Invest 66:892-900).

[0015] Another useful variant of apoA-1 is the Paris variant, where the arginine 151 is replaced with a cysteine.

[0016] The systemic infusion of ApoA-1 alone (Miyazaki et al. 1995, Arterioscler Thromb Vase Biol. 15: 1882-1888 or of HDL (Badimon et al, 1989, Lab Invest. 60:455-461 and J Clin Invest. 85: 1234-1241, 1990) in experimental animals and initial human clinical studies (Nanjee et al., 1999, Arterioscler Thromb Vase Biol. 19:979-989 and Eriksson et al. 1999, Circulation 100:594-598) has been shown to exert significant biochemical changes, as well as to reduce the extent and severity of atherosclerotic lesions.

[0017] Human gene therapy may provide a superior approach for achieving plaque reduction by providing prolonged and continuous expression of genes such as apoA-1 Milano. In the case of conventional gene therapy approaches that add back the entire apoA-1 cDNA, un-regulated expression of this cDNA may lead to toxicity. These problems could be overcome by utilization of spliceosome mediated RNA trans-splicing to convert the wild type apoA-1, or albumin, into Milano or other useful apoA-1 variants.

[0018] Similarly, spliceosome mediated RNA trans-splicing may be used to

simultaneously reduce the expression of apoB, a major component of low-density lipoprotein, and produce HDL, i.e., express apoA-1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function. [0019] Accordingly, a continuing and unmet need exists for new, improved, safer, and alternative means for expression of apoA-1 wild type or variants thereof. This invention addresses these and other needs by providing compositions and methods directed to recombinant lentiviral vectors expressing pre-trans-splicing molecules (PTMs) designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) that is abundantly expressed, and mediate a trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (chimeric RNA) capable of encoding a protein or polypeptide of interest such as apoA-1 wild type or a variant thereof.

[0020] Citation of the above documents or any references cited herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

SUMMARY OF THE INVENTION

[0021] The present invention relates to compositions and methods for generating novel nucleic acid molecules through lentiviral-expressed spliceosome-mediated targeted RNA trans-splicing. The compositions of the invention include lentiviral-expressed pre-trans- splicing molecules (hereinafter referred to as "PTMs") designed to interact with a natural target pre-mRNA molecule (hereinafter referred to as "pre-mRNA") and mediate a spliceosomal trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (hereinafter referred to as "chimeric RNA"). The methods of the invention encompass contacting the lentiviral-expressed PTMs of the invention with a natural target pre-mRNA under conditions in which a portion of the lentiviral-expressed PTM is spliced to the natural pre-mRNA to form a novel chimeric RNA. The lentiviral-expressed PTMs of the invention are genetically engineered so that the novel chimeric RNA resulting from the trans- splicing reaction may encode a protein that provides health benefits. Generally, the target pre- mRNA is chosen because it is expressed within a specific cell type thereby providing a means for targeting expression of the novel chimeric RNA to a selected cell type. For example, lentiviral-expressed PTMs may be targeted to pre-mRNAs expressed in the liver such as apoA-1 and/or albumin pre-mRNA.

[0022] In particular, the compositions of the invention include lentiviral-expressed pre- trans-splicing molecules (hereinafter referred to as "PTMs") designed to interact with an apoA-1 target pre-mRNA molecule (hereinafter referred to as "apoA-1 pre-mRNA") and mediate a spliceosomal trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (hereinafter referred to as "chimeric RNA").

[0023] The compositions of the invention further include lentiviral-expressed PTMs designed to interact with albumin target pre-mRNA molecule (hereinafter referred to as "albumin pre-mRNA") and mediate a spliceosomal trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule.

[0024] The compositions of the invention further include lentiviral-expressed PTMs designed to interact with an apoB target pre-mRNA molecule (hereinafter referred to as "apoB pre-mRNA") and mediate a spliceosomal trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule.

[0025] The compositions of the invention include lentiviral-expressed PTMs designed to interact with an apoA-1 target pre-mRNA molecule, albumin target pre-mRNA, or an apoB target pre-mRNA or other pre-mRNA targets and mediate a spliceosomal trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule. Such lentiviral- expressed PTMs are designed to produce an apoA- 1 or other apoA- 1 variants including Milano which are useful to protect against atherosclerosis.

[0026] The general design, construction and genetic engineering of PTMs and demonstration of their ability to successfully mediate trans-splicing reactions within the cell are described in detail in U.S. Pat. Nos. 6,083,702, 6,013,487 and 6,280,978 as well as U.S. Patent Appl. Ser. Nos. 09/756,095, 09/756,096, 09/756,097 09/941,492, US Pat. Publ. Nos. US 2006-0234247 Al, and US 2006-0194317 Al, the disclosures of which are incorporated by reference in their entirety herein.

[0027] The general design, construction and genetic engineering of trans-splicing ribozymes and demonstration of their ability to successfully mediate trans-splicing reactions within the cell are described in detail in and U.S. Pat. Nos. 5,667,969, 5,854,038 and 5,869,254, as well as U.S. Pat. Publ. Serial No. 20030036517, the disclosures of which are incorporated by reference in their entirety herein.

[0028] The design, construction and genetic engineering of PTMs expressing apoA- 1 or other apoA-1 variants, and highly abundant expressed precursor messenger RNA molecules are described in detail in U.S. Pat. Appl. Nos. 11/141,447 and 11/245,907, respectively, the disclosures of which are incorporated by reference in their entirety herein.

[0029] The methods of the invention encompass contacting the PTMs of the invention with an apoA- 1 target pre-mRNA, albumin target pre-mRNA, or apoB target pre-mRNA, or other expressed pre-mRNA targets, under conditions in which a portion of the PTM is spliced to the target pre-mRNA to form a novel chimeric RNA. The methods of the invention comprise contacting the PTMs of the invention with a cell expressing an apoA- 1 target pre- mRNA, or an apoB target pre-mRNA or other expressed pre-mRNA targets, such as albumin pre-mRNA, under conditions in which the PTM is taken up by the cell and a portion of the PTM is trans-spliced to a portion of the target pre-mRNA to form a novel chimeric RNA molecule that results in expression of the an apoA-1 Milano or another variant. Alternatively, for example, when targeting the albumin or apoB pre-mRNAs, the novel chimeric RNA may encode a wild type apoA-1 protein.

[0030] Alternatively, nucleic acid molecules encoding the PTMs of the invention may be delivered into a target cell followed by expression of the nucleic acid molecule to form a PTM capable of mediating a trans-splicing reaction. The PTMs of the invention are genetically engineered so that the novel chimeric RNA resulting from the trans-splicing reaction may encode the apoA-1 Milano variant protein which has been shown to reduce plaque buildup which may be useful in the prevention or treatment of vascular disease.

Alternatively, the chimeric mRNA may encode a wild type apoA-1 protein. Thus, the methods and compositions of the invention can be used in gene therapy for the prevention and treatment of vascular disorders resulting from accumulation of plaque which is a risk factor associated with heart attacks and strokes.

[0031] In one embodiment, for each of the aforementioned compositions (and methods) of the present invention, the PTMs expressing the apoA-1 or other apoA-1 variants are introduced into the cells using, for example, and not by way of limitation, retroviral vectors, lentiviral vectors, adeno-associated viral based vectors, adenoviral vectors, viral vector transduction, electroporation, transformation, transduction, conjugation, transfection, infection, membrane fusion with cationic lipids, high- velocity bombardment with DNA- coated microprojectiles, incubation with calcium phosphate-DNA precipitate, or direct microinjection into single cells.

[0032] In another preferred embodiment, for each of the aforementioned compositions (and methods) of the present invention, the PTMs expressing the apoA-1 or other apoA-1 variants are introduced into the cells using, for example, certain lentiviral vector constructs including, for example, and not by way of limitation, integration deficient LV, self- inactivating LV, adenovirus-LV hybrids; adeno-associated virus-LV hybrids, and/or combinations thereof. [0033] In yet another aspect of the present invention, the invention provides for a packaging cell line and method of making a packaging cell line for making the LV PTM constructs of the present invention. In one embodiment, a method of producing a

recombinant lentiviral packaging cell is provided comprising introducing into a cell, a nucleic acid capable of expressing in said packaging cell, a nucleic acid sequence to produce transduction-competent virus-like particles; and at least one nucleic acid molecule capable of expressing the sequence of interest in said packaging cell, wherein said packaging cell produces transduction-competent virus-like particles expressing the nucleic acid sequence of interest.

[0034] In each of the aforementioned lentiviral vectors, pharmaceutical compositions containing such lentiviral vectors expressing the PTM constructs of the present invention, and methods of using such lentiviral vectors, the lentiviral vector further comprises one or more of the following including, for example, and not by way of limitation, a nucleic acid sequence encoding functionally active lentiviral RNA packaging elements, a nucleic acid sequence encoding functional central polypurine tract (cPPT), a central termination sequence (CTS) and 3' LTR proximal polypurine tract (PPT), and/or a nucleic acid sequence encoding a nonprotein or protein based marker or tag. In specific embodiments, the lentiviral vector of the present invention comprises one or more of the lentiviral vector constructs depicted in Figure 3, or Figure 7, or any combination thereof.

[0035] In another embodiment, the expression of ApoA- 1 or a variant thereof using at least one PTM encoding ApoA- 1 or a variant thereof, may be used in combination with a therapeutic product(s), which upon irans-splicing, produces a functional ApoA-1 or a variant thereof that causes expression of the therapeutic product(s) within the same cell.

[0036] In another embodiment of the present invention, the at least one first PTM and the at least one second PTM encoding a therapeutic product(s) are only functional after trans- splicing are co-expressed from the same vector, which upon irans-splicing via SMaRT™, cause expression of apoA- 1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function.

[0037] In yet another embodiment of the present invention, the at least one first PTM and the at least one second PTM encoding a therapeutic product(s) are expressed from separate vectors delivered either separately in any order or at the same time, which upon trans- splicing via SMaRT™, cause expression of apoA-1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function. [0038] In yet another embodiment, a cell is provided comprising a recombinant lentiviral vector wherein said lentiviral vector expresses a nucleic acid molecule comprising: a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre- mRNAs expressed within the cell; b) a 3' splice region comprising a branch point and a 3' splice acceptor site; c) a spacer region that separates the 3' splice region from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes an apoAI polypeptide wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

[0039] In yet another embodiment, a method is provided for producing a chimeric RNA molecule in a cell comprising: contacting a target pre-mRNAs expressed in the cell with a nucleic acid molecule expressed by a lentiviral vector, wherein the nucleic acid molecule is recognized by nuclear splicing components, and wherein said nucleic acid molecule comprises: a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNAs expressed within the cell; b) a 3' splice region comprising a branch point and a 3' splice acceptor site; c) a spacer region that separates the 3' splice region from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes an apoAI polypeptide; under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric RNA within the cell.

[0040] In yet another embodiment, a recombinant lentiviral vector is provided wherein said recombinant lentiviral vector expresses a nucleic acid molecule comprising: a) one or more target binding domains that target binding of the nucleic acid molecule to target pre- mRNAs expressed within a cell; b) a 3' splice region comprising a branch point and a 3' splice acceptor site; c) a spacer region that separates the 3' splice region from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes an apoAI polypeptide; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

[0041] In yet another embodiment, a method is provided for expressing an apoAI in a subject comprising administering to said subject a recombinant lentiviral vector wherein said lentiviral vector expresses a nucleic acid molecule comprising: a) one or more target binding domains that target binding of the nucleic acid molecule to target pre-mRNAs expressed within a cell; and b) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes an apoAI polypeptide; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

[0042] In each of the aforementioned embodiments of the methods of the present invention, the irans-splicing is mediated by SMaRT™. In another embodiment, the trans- splicing is mediated by Group I ribozymes. In yet another embodiment, the irans-splicing is mediated by Group II ribozymes.

[0043] In each of the aforementioned embodiments of the compositions and methods of the present invention can comprise pre-trans-splicing molecules (PTMs) that target a highly abundant or expressed pre-mRNAs such as, for example, and not by way of limitation, casein, myosin and fibroin, tumor- specific or tumor associated transcripts, microbial or autoantigen associated transcripts, viral or yeast associated transcripts, and contain the coding sequence of a protein or polypeptide of interest for example, and not by way of limitation, Factor VIII protein, cytokines, growth factors, insulin, hormones, enzymes and antibody polypeptides.

[0044] These and other aspects of some exemplary embodiments will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments without departing from the spirit thereof. Additional features may be understood by referring to the accompanying drawings, which should be read in conjunction with the following detailed description and examples.

BRIEF DESCRIPTION OF THE FIGURES

[0045] FIG. 1 schematically illustrates the use of SMaRT™ to mediate trans-splicing into Albumin.

[0046] FIG. 2 schematically illustrates the Pre-Trans-splicing Molecules (PTMs).

[0047] FIG. 3 schematically illustrates the LV-hApoA-1 PTM cassette.

[0048] FIG. 4 schematically illustrates the therapeutic use of SMaRT™ technology to test lentiviral expression of hApoA-I PTM in HepG2.

[0049] FIG. 5 schematically illustrates the therapeutic use of SMaRT™ technology in treatment of Trans-splicing in primary human hepatocytes with VRX1243.

[0050] FIG. 6 schematically illustrates unspliced PTM RNA expression from different MuAlb-HuApaA-1 PTMs. [0051] FIG. 7 schematically illustrates lentiviral vector configurations for the LV PTM constructs.

[0052] The maps illustrated in the drawings are not drawn to scale, and the relative sizes of particular segments or functional elements are not necessarily proportional to the lengths (e.g. , number of base pairs) of the corresponding sequences.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0053] As used herein, each of the following terms has the meaning associated with it in this section.

[0054] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0055] As used herein, the term "vector" means a nucleic acid molecule (typically DNA or RNA) that serves to transfer a passenger nucleic acid sequence (i.e., DNA or RNA) into a host cell. Three common types of vectors include plasmids, phages and viruses. Preferably, the vector is a virus, which includes the encapsidated forms of vector nucleic acids, and viral particles in which the vector nucleic acids have been packaged. Transduction of cells with LV includes, but not limited to, three major steps: cell entry, conversion of vector RNA into DNA and delivering of DNA to nucleus. Transduction-competent (or maybe capable) LVs have all elements to accomplish all above-mentioned steps.

[0056] As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/ regulatory sequence may, for example, be one which expresses the gene product in a tissue-specific manner.

[0057] As used herein, a "tissue-specific" promoter means a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. Pre-Trans-Splicing Molecules (PTMs) Encoding ApoA-1 or Variants Thereof

[0058] The present invention relates to compositions and methods for generating novel nucleic acid molecules through lentiviral-expressed spliceosome-mediated targeted RNA trans-splicing. The compositions of the invention include lentiviral-expressed pre-trans- splicing molecules (hereinafter referred to as "PTMs") designed to interact with a natural target pre-mRNA molecule (hereinafter referred to as "pre-mRNA") and mediate a spliceosomal trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (hereinafter referred to as "chimeric RNA"). The methods of the invention encompass contacting the lentiviral-expressed PTMs of the invention with a natural target pre-mRNA under conditions in which a portion of the lentiviral-expressed PTM is spliced to the natural pre-mRNA to form a novel chimeric RNA. The lentiviral-expressed PTMs of the invention are genetically engineered so that the novel chimeric RNA resulting from the trans- splicing reaction may encode a protein that provides health benefits. Generally, the target pre- mRNA is chosen because it is expressed within a specific cell type thereby providing a means for targeting expression of the novel chimeric RNA to a selected cell type. For example, lentiviral-expressed PTMs may be targeted to pre-mRNAs expressed in the liver such as apoA-1 and/or albumin pre-mRNA.

[0059] In each embodiment of the compositions of the aforementioned lentiviral expressed ApoA- 1 PTMs of the present invention and methods of using same as described in detail herein, the ApoA-1 encoded by the at least one PTM also specifically includes those derivatives, fragments or modifications thereof, which upon irans-splicing, cause expression of ApoA-1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations of the ApoA- 1 PTMs described herein that are the result of natural genotypic, allelic variation, or that have been artificially engineered, and which, upon irans-splicing, cause expression of apoA-1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function, are intended to be within the scope of the invention.

[0060] Thus, derivatives, fragments or modifications thereof of the ApoA-1 PTMs encoded by the at least one PTM can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the pluripotency factor(s), such that one or more amino acid residue substitutions, additions, or deletions are introduced into the ApoA-1 PTMs encoded by the at least one PTM. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted nonessential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence of the ApoA-1 PTMs, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that, upon irans-splicing using SMaRT™, cause expression of apoA-1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA-1- Milano function.

[0061] Now referring specifically to the attached drawings, in one embodiment of the present invention, a schematic representation of gene correction or therapeutic protein expression using PTM's targeted to ApoA-1 is depicted in FIG. 1. The PTMs coding for ApoA-1 and, optionally, PTM's to correct the gene defect or to express a therapeutic protein (PTM set 2) are introduced into the cells using, for example, and not by way of limitation, retroviral vectors, lentiviral vectors, adeno-associated viral based vectors, adenoviral vectors, viral vector transduction, electroporation, transformation, transduction, conjugation, transfection, infection, membrane fusion with cationic lipids, high- velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitate, or direct microinjection into single cells. The ApoA-1 PTM set 1 is targeted to endogenous pre- mRNAs that are expressed in the dividing or non-dividing somatic cells, and following trans- splicing, cause expression of apoA- 1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function.

[0062] The PTMs of the invention comprise a target binding domain that is designed to specifically bind to endogenous pre-mRNA, a 3' splice region that includes a branch point, pyrimidine tract and a 3' splice acceptor site and/or a 5' splice donor site; and a spacer region that separates the RNA splice site from the target binding domain. In addition, the PTMs of the invention can be engineered to contain any nucleotide sequences encoding a ApoA- 1 PTM, which upon irans-splicing, cause expression of apoA-1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function. In a preferred embodiment, the ApoA- 1 PTM translated upon irans-splicing using SMaRT™ cause expression of apoA- 1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function. The methods of the invention encompass contacting the PTMs of the invention with a natural endogenous pre-mRNA under conditions in which a portion of the PTM is irans-spliced to a portion of the natural endogenous pre-mRNA to form a novel chimeric mRNA. Specificity can be achieved by modification of the binding domain of the PTM to bind to the target endogenous pre-mRNA.

[0063] The PTMs of the invention thus comprise (i) one or more target binding domains that target binding of the PTM to a pre-mRNA (ii) a 3' splice region that includes a branch point, pyrimidine tract and a 3' splice acceptor site and/or 5' splice donor site; and (iii) a spacer region to separate the RNA splice site from the target binding domain. Additionally, as described above, the PTMs are engineered to contain any nucleotide sequence encoding a ApoA-1, which upon irans-splicing, cause expression of apoA-1 wild type or the Milano variant or convert other expressed proteins such as albumin to produce ApoA- 1 -Milano function.

[0064] The target binding domain of the PTM may contain one or two binding domains of at least 15 to 30; or having long binding domains as described in US Patent Publication No. US 2006-0194317 Al (the contents of which are incorporated herein by reference in their entirety), of up to several hundred nucleotides which are complementary to and in anti-sense orientation to the targeted region of the selected endogenous pre-mRNA. This confers specificity of binding and anchors the endogenous pre-mRNA closely in space so that the spliceosome processing machinery of the nucleus can irans-splice a portion of the PTM to a portion of the endogenous pre-mRNA. A second target binding region may be placed at the 3' end of the molecule and can be incorporated into the PTM of the invention. Absolute complementarity, although preferred, is not required. A sequence "complementary" to a portion of the endogenous pre-mRNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the endogenous pre-mRNA, forming a stable duplex. The ability to hybridize will depend on both the degree of complementarity and the length of the nucleic acid (See, for example, Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ν.Υ.)· Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex. One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

[0065] Binding may also be achieved through other mechanisms, for example, through triple helix formation or protein/ nucleic acid interactions such as those in which the PTM is engineered to recognize a specific RNA binding protein, i.e., a protein bound to a specific target endogenous pre-mRNA. Alternatively, the PTMs of the invention may be designed to recognize secondary structures, such as for example, hairpin structures resulting from intramolecular base pairing between nucleotides within an RNA molecule.

[0066] The PTM molecule also contain a 3' splice region that includes a branch point, pyrimidine tract and a 3' splice acceptor AG site and/or a 5' splice donor site. Consensus sequences for the 5' splice donor site and the 3' splice region used in RNA splicing are well known in the art (See, Moore, et al., 1993, The RNA World, Cold Spring Harbor Laboratory Press, p. 303-358). In addition, modified consensus sequences that maintain the ability to function as 5' donor splice sites and 3' splice regions may be used in the practice of the invention. Briefly, the 5' splice site consensus sequence is AG/GURAGU (where A = adenosine, U = uracil, G = guanine, C = cytosine, R = purine and / = the splice site). The 3' splice site consists of three separate sequence elements: the branch point or branch site, a polypyrimidine tract and the 3' consensus sequence (YAG). The branch point consensus sequence in mammals is YNYURAC (Y = pyrimidine). The underlined A is the site of branch formation. A polypyrimidine tract is located between the branch point and the splice site acceptor and is important for branch point utilization and 3' splice site recognition.

[0067] A spacer region to separate the RNA splice site from the target binding domain is also included in the PTM. The spacer region can have features such as stop codons which would block any translation of an unspliced PTM and/or sequences that enhance trans- splicing to the target pre-mRNA.

[0068] In a preferred embodiment of the invention, a "safety" design of the binding domain is also incorporated into the spacer, binding domain, or elsewhere in the PTM to prevent non-specific irans-splicing. The spacer sequence is a region of the PTM that covers elements of the 3' and/or 5' splice site of the PTM by relatively weak complementarity thereby preventing non-specific irans-splicing. The PTM is designed in such a way that upon hybridization of the binding/targeting portions) of the PTM, the 3' and/or 5'splice site is uncovered and becomes fully active. The "safety" sequence consists of one or more complementary stretches of cis-sequence (or could be a second, separate, strand of nucleic acid) which weakly binds to one or both sides of the PTM branch point, pyrimidine tract, and/or 3' splice site (splicing elements), or could bind to parts of the splicing elements themselves. This "safety" sequence binding prevents the splicing elements from being active (i.e. block U2 snRNP or other splicing factors from attaching to the PTM splice site recognition elements). The binding of the "safety" sequence may be disrupted by the binding of the target binding region of the PTM to the target pre-mRNA, thus exposing and activating the PTM splicing elements (making them available to trans- splice into the target endogenous pre-mRNA).

[0069] Additional features can be added to the PTM molecule either after, or before, the nucleotide sequence encoding a translatable protein, such as polyadenylation signals or 5' splice sequences to enhance splicing, additional binding regions, "safety" sequence self- complementary regions, additional splice sites, or protective groups to modulate the stability of the molecule and prevent degradation. Additional features that may be incorporated into the PTMs of the invention include stop codons or other elements in the region between the binding domain and the splice site to prevent unspliced pre-mRNA expression. In another embodiment of the invention, PTMs can be generated with a second anti-sense binding domain downstream from the nucleotide sequences encoding a translatable protein to promote binding to the 3' target intron or exon and to block the fixed authentic cis-5' splice site (U5 and/or Ul binding sites). PTMs may also be made that require a double trans- splicing reaction for expression of the trans- spliced product. Such PTMs could be used to replace an internal exon which could be useful for RNA repair. Further elements such as a 3' hairpin structure, circularized RNA, nucleotide base modification, or a synthetic analog can be incorporated into PTMs to promote or facilitate nuclear localization and spliceosomal incorporation, and intracellular stability.

[0070] The PTMs of the invention can be used in methods designed to produce a novel chimeric mRNA in a target cell such as, for example, a somatic cell. The methods of the present invention comprise delivering to the target cell a PTM which may be in any form used by one skilled in the art, for example, an RNA molecule, an RNA vector or a DNA vector which is transcribed into a RNA molecule, wherein the PTM binds to an endogenous pre-mRNA and mediates a trans- splicing reaction resulting in formation of a chimeric RNA comprising a portion of the PTM molecule spliced to a portion of the endogenous pre- mRNA.

[0071] The PTMs of the present invention can be delivered using viral vectors (e.g., lentiviral, Adeno-associated viral ("AAV"), Adenoviral, EBV, HSV, Rabies, hybrid vectors comprising AAV and Lentiviral vector, etc.) or non-viral vectors (e.g. plasmid DNA vectors including, for example, minicircle DNA vectors, (Chen et ah , Hum Gene Ther 16, 126-131, 2005), transposon delivery systems, phage, or PTM RNA molecules. FIG. 3 shows a non- limiting example of an LV PTM construct of the present invention. Furthermore, the expression of the PTMs can be regulated by a constitutive promoter(s) or an inducible promoter(s) or a tissue specific promoter(s) or their combination, and may be bidirectional, capable of driving the expression of one or more different PTMs in a single vector (FIG. 7).

[0072] Thus, the present invention describes the use of SMaRT™ technology to produce ApoA-1 or variants thereof in patient specific somatic cells. This is achieved by trans- splicing PTMs encoding ApoA-1 or variants thereof into one or more endogenous pre- mRNAs in somatic cells. The target pre-mRNA transcripts can include those that are constitutively expressed or that are up or down regulated.

[0073] The PTMs can therefore be designed with different binding domains and coding domains to target defective pre-mRNA for repair or to modify highly expressed pre-mRNAs to generate therapeutic proteins of interest or image gene expression for diagnostic applications. Trans-splicing between the PTM and target pre-mRNA may not occur until the pre-mRNA is expressed, which may be critical for some applications where early expression of a transgene may be detrimental to the cell, e.g. expression of cystic fibrosis transmembrane conductance regulator protein (CFTR) in pulmonary stem cells.

[0074] Alternatively, the genes or PTMs can be excised, e.g. by incorporating Lox-sites into integrating vectors and expressing Cre-recombinase, or silenced, e.g. by incorporating sequence(s) targeted by stage (lineage-, tissue-)-specific siRNA or micro-RNA, as an additional safety measure.

Lentiviral vectors

[0075] The LV-PTM constructs of the present invention comprise a 5 ' LTR and a 3 ' LTR; a first nucleic acid sequence operably linked to said 5' LTR, also referred to herein as the "pay load"; and a second nucleic acid sequence, that is operably linked to said 5' LTR wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR. "Pay load" is that portion of the vector which is distinct from the packaging signal required to package the RNA version of the lentiviral vector during viral production. In certain embodiments, a minimum packaging sequence may be used.

[0076] The vector of the present invention further comprises a nucleic acid sequence encoding functionally active lentiviral RNA packaging elements. The full-length lentiviral RNA is selectively incorporated into the viral particles as a noncovalent dimer. RNA packaging into virus particles is dependent upon specific interactions between RNA and the nucleocapsid protein (NC) domain of the Gag protein. In nature, incorporation of the HIV genomic RNA into the viral capsid (referred to as "encapsidation") involves the so-called Psi region located immediately upstream of the Gag start codon and folded into four stem-loop structures, is important for genome packaging; SL1 to SL4. In particular, SL1 contains the dimerization initiation site (DIS), a GC-rich loop that mediates in vitro RNA dimerization through kissing-complex formation, presumably a prerequisite for virion packaging of RNA. Additional cis-acting sequences have also been shown to contribute to RNA packaging. Some of these elements are located in the first 50 nucleotides (nt) of the Gag gene, including SL4, whereas others are located upstream of the splice-donor site (SD1), and are actually mapped to a larger region covering the first 350-400 nt of the genome, including about 240 nt upstream of SL1. The SL1-4 region is an example of a simple sequence essential for RNA packaging. Other such sequences are known by those of skill in the art.

[0077] The LV-PTM constructs also comprise a nucleic acid sequence encoding a functional central polypurine tract (cPPT)/cTS and 3' LTR proximal polypurine tract (PPT). HIV and other lentiviruses, as are known in the art, have the unique property to replicate in non-dividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap; the central DNA flap. This central DNA flap is a region of triplestranded DNA created by two discrete half-genomic fragments with a central strand displacement event controlled in cis by a central polypurine tract (cPPT) and a central termination sequence (CTS) during HIV reverse transcription. A central copy of the polypurine tract ds-active sequence (cPPT), present in all lentiviral genomes, initiates synthesis of a downstream plus strand. The upstream plus strand segment initiated at the 3 ' PPT will, after a strand transfer, proceed until the center of the genome and terminate after a discrete strand displacement event. This last event of HIV reverse transcription is controlled by the central termination sequence (CTS). [0078] It is an aspect of the present invention that the transcription of the payload is driven by the 5' LTR. The 5' LTR has sufficient basal activity to drive transcription of a payload comprising nucleic acids that encode full length antigenic sequences, as well as packaging sequences. The 5' LTR can be derived from various strains and clades of HIV, as are known in the art, and optimized for stronger basal promoter-like function. In particular, the 5' LTR from HIV-1 Clade E can exhibit strong basal promoter activity. Various strains and clades of HIV are known in the art and may be used to generate the lentiviral vaccine vectors of the present invention including for example, without limitation, HIV-1 groups: M (for major)(A, B, C, D, E, F, G, H, I, and J), O (outlier or "outgroup"), which is a relatively rare group currently found in Cameroon, Gabon, and France, and a third group, designated N (new group), and any circulating recombinant forms thereof. The 5' LTR further drives expression of the payload. The HIV Rev protein directs the export of unspliced or partially spliced viral transcripts from the nucleus to the cytoplasm in mammalian cells. Rev contains the RNA binding domain, which binds the RRE present on target transcripts. Export activity is mediated by a genetically defined effector domain, which has been identified as a nuclear export signal.

[0079] In another embodiment of the present invention, the LV-PTM constructs of the present invention can comprise at least one, but can optionally comprise two or more nucleotide sequences of interest (second PTM, third PTM, etc.). In order for two or more nucleotide sequences of interest to be expressed, there may be two or more transcription units within the vector genome, one for each nucleotide sequences of interest. In those instances, it is preferable to use one or more internal ribosome entry sites (IRESs) or FMDV 2A-like sequences for translation of the second (and subsequent) coding sequence(s) in a poly- cistronic (or as used herein, "multicistronic") message (Adam et al 1991 J. Virol. 65, 4985, the entire contents of which are incorporated herein by reference). The IRES/2 A(s) may be of viral origin (such as EMCV IRES, PV IRES, or FMDV 2A-like the entire contents of which are incorporated herein by reference sequences) or cellular origin (such as FGF2 IRES, NRF IRES, Notch 2 IRES or EIF4 IRES). Non-limiting examples of lentiviral vector constructs of the present invention that utilize an IRES sequence may be found in Figure 7, infra.

[0080] In addition, in certain embodiments of the present invention, the second nucleotide sequence of interest or "payload" sequence can also includes those nucleotide sequences encoding enzymes, cytokines, chemokines, growth factors, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, immune co- stimulatory molecules, immunomodulatory molecules, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumour suppresser protein and growth factors, membrane proteins, pro- and anti- angiogenic proteins and peptides, vasoactive proteins and peptides, anti-viral proteins and derivatives thereof (such as with an associated reporter group). The nucleotide sequences of interest may also encode pro-drug activating enzymes. When used in a research context, the nucleotide sequences of interest may also encode reporter genes such as, but not limited to, green fluorescent protein (GFP), luciferase, .beta.-galactosidase, or resistance genes to antibiotics such as, for example, ampicillin, neomycin, bleomycin, zeocin, chloramphenicol, hygromycin, kanamycin, among others. The nucleotide sequences of interest may also include those which function as anti-sense RNA, small interfering RNA (siRNA), or ribozymes, or any combination thereof.

[0081] In another embodiment of the present invention, the lentiviral vector of the LV- PTM of the present invention may include, without limitation, those lentiviruses can be divided into viruses that infect primate (HIV-1, HIV-2, simian immunodeficiency virus (SIV)) and non-primate (feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), Bovine Immunodeficiency Virus (BIV), caprine arthritis encephalitis virus (CAEV), visna maedi virus (VV), Jembrana disease virus (JDV)).

[0082] In yet another embodiment of the present invention, the lentiviral vector of the present invention could be also modified by removing the transcriptional elements of HIV LTR; such as in a so-called self-inactivating (SIN) vector configuration. The modalities of reverse transcription, which generates both U3 regions of an integrated pro virus from the 3' end of the viral genome, facilitate this task by allowing the creation of so-called self- inactivating (SIN) vectors. Self-inactivation relies on the introduction of a disruption (employing for example, deletion, mutation and element insertion) in the U3 region of the 3 ' long terminal repeat (LTR) of the DNA used to produce the vector RNA. During reverse transcription, this deletion is transferred to the 5' LTR of the pro viral DNA. If enough sequence is eliminated to abolish the transcriptional activity of the LTR, the production of full-length vector RNA in transduced cells is abolished. This minimizes the risk that replication competent lentiviruses (RCLs) will emerge. Furthermore, it reduces the likelihood that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed, either due to the promoter activity of the 3 ' LTR or through an enhancer effect. Finally, a potential transcriptional interference between the LTR and the internal promoter driving the transgene is prevented by the SIN design. One example of a SIN based lentiviral vector is described in U.S. Pat. No. 6,924,144, the entire contents of which are incorporated herein by reference in its entirety. Non-limiting representative examples of SIN-based lentiviral vectors of the present invention may be generated from one or more of the constructs specifically shown in Figure 7 described herein or any combination thereof.

[0083] In yet another embodiment, the lentiviral vector of the present invention could be also modified so that the left or right or both LTRs of the LV-PTM construct of the present invention contain one or more insulator element(s). Non-limiting examples of insulator sequences may be those based upon the .alpha. -globin locus, including, for example, chicken HS4 such as disclosed in U.S. Pat. Publ. No. 0057725, the entire contents of which are incorporated herein by reference).

[0084] In yet another embodiment, the invention includes a pharmaceutical composition comprising the LV-PTM construct described herein above comprising: a 5' LTR and a 3' LTR; a first nucleic acid sequence operably linked to said 5' LTR; and a second nucleic acid sequence operably linked to said 5' LTR, wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR; and further comprising a "pharmaceutically acceptable carrier" or "genetic adjuvant." "Pharmaceutically acceptable carriers" include, without limitation, PBS, buffers, water, TRIS, other isotonic solutions or any solution optimized to not damage the viral components of the vector.

[0085] The above described lentiviral vectors can be introduced into a host cell for the therapeutic treatment of diseases, as well as for other reasons described herein. Accordingly, the present invention provides a host cell comprising a vector according to the invention. The isolation of host cells, and/or the maintenance of such cells or cell lines derived therefrom in culture, has become a routine matter and one in which the ordinary skilled artisan is well versed.

[0086] A "host cell" can be any cell, and, preferably, is a eukaryotic cell. Desirably, the host cell is an antigen presenting cell. Such a cell includes, but is not limited to, a skin fibroblast, a bowel epithelial cell, an endothelial cell, an epithelial cell, a dendritic cell, a plasmacytoid dendritic cell, Langerhan's cells, a monocyte, a mucosal cell, a liver cell (a perenchymal cell such as a hepatocyte), or a non-parenchymal cell (an endothelial cell, a kupffer cell, a stellate cell, oval cell), or any of the precursors thereto such as hepatic stem cells, bone marrow liver stem cells, and the like. Preferably, the host cell is of a eukaryotic, multicellular species (e.g. , as opposed to a unicellular yeast cell), and, even more preferably, is a mammalian, e.g. , human cell.

[0087] A cell can be present as a single entity, or can be part of a larger collection of cells. Such a "larger collection of cells" can comprise, for instance, a cell culture (either mixed or pure), a tissue (e.g. , endothelial, epithelial, mucosa or other tissue), an organ (e.g. , lung, liver, muscle and other organs), an organ system (e.g. , circulatory system, respiratory system, gastrointestinal system, urinary system, nervous system, integumentary system or other organ system), or an organism (e.g. , a bird, mammal, or the like). Preferably, the organs/tissues/cells being targeted are of the circulatory system (e.g. , including, but not limited to blood, including white blood cells), the mucosal system of the nose, trachea, bronchi, bronchioles, lungs, and the like), gastrointestinal system (e.g. , including mouth, pharynx, esophagus, stomach, intestines, salivary glands, pancreas, liver, gallbladder, and others), urinary system (e.g. , such as kidneys, ureters, urinary bladder, urethra, and the like), nervous system (e.g. , including, but not limited to, brain and spinal cord, and special sense organs, such as the eye) and integumentary system (e.g. , skin, epidermis, and cells of subcutaneous or dermal tissue). Even more preferably, the cells being targeted are selected from the group consisting of antigen presenting cells. The target cells need not be normal cells and can be diseased cells. Such diseased cells can be, but are not limited to, tumor cells, infected cells, genetically abnormal cells, or cells in proximity or contact to abnormal tissue such as tumor vascular endothelial cells.

[0088] A "vector" is a nucleic acid molecule (typically DNA or RNA) that serves to transfer a passenger nucleic acid sequence (i.e., DNA or RNA) into a host cell. Three common types of vectors include plasmids, phages and viruses. Preferably, the vector is a virus, which includes the encapsidated forms of vector nucleic acids, and viral particles in which the vector nucleic acids have been packaged. Transduction of cells with LV includes, but not limited to, three major steps: cell entry, conversion of vector RNA into DNA and delivering of DNA to nucleus. Transduction-competent (or maybe capable) LVs have all elements to accomplish all above-mentioned steps.

[0089] The vector is not a wild-type strain of a virus, inasmuch as it comprises human- made mutations or modifications. Thus, the vector typically is derived from a wild-type viral strain by genetic manipulation (e.g. , by addition, deletion, mutation, insertion or other techniques known in the art) to comprise lentivirus, as further described herein. As encompassed herein, a vector can comprise either DNA or RNA. For instance, either a DNA or RNA vector can be used to derive the virus. Similarly, a cDNA copy can be made of a viral RNA genome. Alternatively, a cDNA (or viral genomic DNA) moiety can be transcribed in vitro to produce RNA. These techniques are well-known to those skilled in the art, and also are described.

[0090] In yet another aspect of the present invention, a method of propagating and selectively packaging an LV-PTM of the present invention is provided. The method comprises cells, cell lines and methods as are known in the art and more particularly described in commonly owned applications Application Number: 60/585,464, July 1, 2004, Application Number 11/172,147, filed June 30, 2005, US Patent Number 6,835,568, and US Publication Number 20030026791, the text of each of these recited applications, patent, and published application are incorporated herein by reference in their entirety as if fully set forth herein.

[0091] The use of integrating vectors generates a heightened level of safety concern compared to non-integrating delivery systems. In the case of the widely reported X- linked SCID (X-SCID) trials using a Murine leukemia (MLV) retroviral vector it is now thought that insertion of the vector activated an oncogene leading to leukemia in five out of twenty patients. Subsequent studies strongly suggest that the type of transgene and vector, the genetic disorder involved, and the selective advantage gained by cells that were corrected all combined for an unfavorable outcome. There is some evidence that integration of HIV-1- based vectors in hematopoietic cells can perturb the expression of some genes close to the integration site (600 kb), but global gene expression is unaltered. Other mouse models have been developed to assess the safety of Lentiviral vector (LV) and a majority of these studies have shown no association of LV transduction and integration with the development of either replication competent virus (RCL) or increased tumorigenesis.

[0092] Although lentiviral vectors integrate into the host genome, they can be produced as integration defective vectors by disrupting the integrase function of the HIV pol gene. This system will be transient in nature and will be progressively lost as the cells divide thus providing an additional safety layer. Additionally, integration defective vectors will also present much lower risk of insertional mutagenesis and activation or disruption of endogenous genes.

[0093] In yet another embodiment of the present invention, the LV-PTM constructs of the present invention further comprise those lentiviral vectors in which the lentiviral integrase function has been deleted and/or abrogated by site directed mutagenesis. Insertional mutagenesis has been observed in clinical trials with oncoretroviral vectors and this has prompted detailed study of genotoxicty of all integrating vectors. The most straightforward approach for several vaccine applications would be avoiding the possibility of integration. Non-integrating lentiviral vectors have been developed by mutating the integrase gene or by modifying the attachment sequences of the LTRs. In particular, among the mutations studied, the D64V substitution in the catalytic domain has been frequently used because it shows the strong inhibition of the integrase without affecting proviral DNA synthesis. It has been reported that the mutation allows a transduction efficiency only slightly lower than integrative vectors but a residual integration that is about 1000-fold lower than an integrative vector at low vector doses. Another mutation described, Dl 16N, resulted in residual integration about 2000 times lower than control vectors. In a couple of instances it has been shown that a single administration of an integrase (IN) -defective SIN LV elicits a significant immune response in the absence of vector integration and may be a safe and useful strategy for vaccine development. Thus, specifically contemplated within the scope of this invention is the modification to render the lentiviral vectors able to exist in episomal form yet still being able to provide transgene expression.

[0094] The lentiviral vector can be compatible with the host cell into which it is introduced, i.e., it is capable of imparting expression on the cell of the vector-encoded nucleic acid sequences. A coding sequence is "operably linked" to a promoter when the promoter is capable of directing transcription of the coding sequence.

[0095] "Pseudotyping" a virion is accomplished by co-transfecting a packaging cell with both the lentiviral vector of interest and a helper vector encoding at least one envelope protein of another virus or a cell surface molecule (see, for example, U.S. Patent Number 5,512,421, the entire text of which is herein incorporated by reference in its entirety). One viral envelope protein commonly used to pseudotype lentiviral vectors is the vesicular stomatitis virus -glycoprotein G (VSV-G), which is derived from a rhabdovirus. Other viral envelopes proteins that may be used include, for example, rabies virus-glycoprotein G and baculovirus gp-64. The use of pseudotyping broadens the host cell range of the lentiviral vector particle by including elements of the viral entry mechanism of the heterologous virus used. Pseudotyping of lentiviral vectors with, for example, VSV-G for use in the present invention results in lentiviral particles containing the lentiviral vector nucleic acid

encapsulated in a nucleocapsid which is surrounded by a membrane containing the VSV-G envelope protein. The nucleocapsid preferably contains proteins normally associated with the lentiviral vector. The surrounding VSV-G protein containing membrane forms part of the viral particle upon its egress from the producer cell used to package the lentiviral vector. In an embodiment of the invention, the lentiviral particle is derived from HIV and pseudotyped with the VSV-G protein. Pseudotyped lentiviral particles containing the VSV-G protein can infect a diverse array of cell types with higher efficiency than amphotropic viral vectors. The range of host cells includes both mammalian and non-mammalian species, such as humans, rodents, fish, amphibians and insects.

[0096] Even though VSV-G pseudotyping has been described as being the most efficient for cutaneous transduction, a great advantage of using LV is that it is possible to target the vector to specific tissues or cells by replacing and/or modifying the virion envelope. LVs are remarkably compatible with a broad range of viral envelope glycoproteins providing them with added flexibility; Rabies, Mokola, LCMV, Ross River, Ebola, MuLV, Baculovirus GP64, HCV, Sindai virus F protein, Feline Endogenous Retrovirus RD114 modified, Human Endogenous Retroviruses, Seneca virus, GALV modified and HA influenza glycoproteins or a combination thereof, to name a few of those viral envelope glycoproteins explored. In addition to modification or replacement of the entire envelope, flexibility of LV platform for targeting different cell types was further demonstrated by refining the surface of LV particles via the display of cell-specific ligands. For vaccine applications, VSV-G as a pseudotyping envelope confers some important advantages, such as a broad cellular tropism (including dendritic cells) and low preexisting immunity in the human population. VSV-G could eventually be replaced by other envelopes if needed, for example in the case of multiple vector administration, although anti- VSV-G immunity does not seem to prevent repeated vector administrations.

[0097] In certain embodiments of the present invention, the heterologous promoter comprises viral, human, and/or synthetic promoters or a combination thereof. In one embodiment, heterologous viral promoters comprise Mouse Mammary Tumor Virus (MMTV) promoter, Moloney virus, avian leukosis virus (ALV), Cytomegalovirus (CMV) immediate early promoter/enhancer, Rous Sarcoma Virus (RSV), adeno-associated virus (AAV) promoters; adenoviral promoters, and Epstein Barr Virus (EBV) promoters, or any combination thereof. In one embodiment, heterologous human promoters comprise

Apolipoprotein E promoter, Albumin promoter, Human ubiquitin C promoter, human tissue specific promoters such as liver specific promoter (for example, HCR-hATT), prostate specific antigen (psa) promoter, Human phosphoglycerate kinase (PGK) promoter, Elongation factor-1 alpha (EF-la) promoter, dectin-2 promoter, HLA-DR promoter, Human CD4 (hCD4) promoter, or any combination thereof. In yet another embodiment, the synthetic promoters comprise those promoters described in US Patent 6,072,050, the contents of which are incorporated by reference in its entirety.

[0098] It is yet another aspect of the invention that various elements of the construct may include functional equivalent derivatives, fragments or modifications thereof that have been engineered into the nucleotide coding sequences and/or amino acid sequences of the first nucleic acid sequence or the second nucleic acid sequence of the lentiviral vectors of the present invention. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural genotypic, allelic variation, or that have been artificially engineered, and that do not alter the functional activity are intended to be within the scope of the invention. Thus, functional equivalent derivatives, fragments or modifications thereof of the first nucleic acid sequence or the second nucleic acid sequence of the lentiviral vectors of the present invention can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of marker nucleic acids, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined. Methods of Use

[0099] The LV-PTM compositions and methods of the present invention are designed to substitute apoAI, or apoB expression, or other pre-mRNA targets, such as albumin, with wild-type apoAI, apoAI Milano or other apoAI variant expression. Specifically, targeted trans-splicing, including double-trans-splicing reactions, 3' exon replacement and/or 5' exon replacement can be used to substitute apoAI, apoB, or albumin sequences with either wild type apoAI or apoAI Milano sequences resulting in expression of apoAI wild type or Milano variant.

[00100] In addition to the lentiviral vector expressing the PTMs of the present invention, various delivery systems are known and can be used to transfer the compositions of the invention into cells, e.g. encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the composition, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral, adenoviral, adeno-associated viral or other vector, incorporation into a plasmid or mini-circle, injection of DNA, electroporation, calcium phosphate mediated transfection, etc.

[0100] The compositions and methods can be used to provide a gene encoding a wild- type apoAI, apoAI Milano, apoB/apoAI wild type or Milano, alb/apoAI wild type or milano chimeric protein to cells of an individual where expression of said gene products reduces plaque formation.

[0101] Specifically, the compositions and methods can be used to provide sequences encoding a wild type apoAI, an apoAI Milano variant molecule, or apoB/apoAI or alb/apoAI chimeric protein to cells of an individual to reduce the plaque formation normally associated with vascular disorders leading to heart attacks and stroke.

[0102] In a preferred embodiment, nucleic acids comprising a sequence encoding a PTM are administered to promote PTM function, by way of gene delivery and expression into a host cell. In this embodiment of the invention, the nucleic acid mediates an effect by promoting PTM production. Any of the methods for gene delivery into a host cell available in the art can be used according to the present invention. For general reviews of the methods of gene delivery see Strauss, M. and Barranger, J. A., 1997, Concepts in Gene Therapy, by Walter de Gruyter & Co., Berlin; Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol.

33:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; 1993, TIBTECH 11(5): 155-215. Exemplary methods are described below.

[0103] Delivery of the LV-PTM into a host cell may be either direct, in which case the host is directly exposed to the LV-PTM or LV-PTM encoding nucleic acid molecule, or indirect, in which case, host cells are first transformed with the LV-PTM or LV-PTM encoding nucleic acid molecule in vitro or ex vivo, then transplanted into the host. These two approaches are known, respectively, as in vivo or ex vivo gene delivery.

[0104] In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the PTM. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g. by infection using a defective or attenuated retroviral or other viral vector (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont, Bio-Rad), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432).

[0105] Another approach to gene delivery into a cell involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. The resulting recombinant cells can be delivered to a host by various methods known in the art. In a preferred embodiment, the cell used for gene delivery is autologous to the host's cell.

[0106] In a specific embodiment of the invention, hepatic stem cells, oval cells, or hepatocytes may be removed from a subject and transfected with a nucleic acid molecule capable of encoding a PTM designed to produce, upon trans-splicing, a wild-type apoAI, an apoAI Milano or other apoAI variant protein and/or apoB/apoAI or alb/apoAI chimeric protein. Cells may be further selected, using routine methods known to those of skill in the art, for integration of the nucleic acid molecule into the genome thereby providing a stable cell line expressing the PTM of interest. Such cells are then transplanted into the subject, thereby providing a source of wild type apoAI, or apoAI Milano variant protein. [0107] While the LV PTM construct of the present invention has been exemplified using a PTM expressing ApoA-1, and specifically targeting albumin as the highly abundant pre- mRNA transcript, each of the aforementioned embodiments of the compositions and methods of the present invention can comprise pre-trans-splicing molecules (PTMs) that target other highly abundant or expressed pre-mRNAs such as, for example, and not by way of limitation, casein, myosin and fibroin, tumor- specific or tumor associated transcripts, microbial or autoantigen associated transcripts, viral or yeast associated transcripts, or any combination thereof. Similarly, while the LV PTM construct of the present invention has been exemplified using a PTM expressing ApoA-1, specifically targeting albumin as the highly abundant pre- mRNA transcript, the coding sequence of a protein or polypeptide of interest, for example, and not by way of limitation, that may be expressed by the PTM may include Factor VIII protein, cytokines, growth factors, insulin, hormones, enzymes, antibody polypeptides, or any combination thereof.

[0108] By way of illustration, and not by way of limitation, in the case of a LV PTM FVIII construct of the present invention, the PTM construct would be genetically engineered so as to lack the native translation initiation codon and the FVIII signal peptide that is required for secretion. Accordingly, the LV-PTM construct would not contain the complete FVIII coding sequences of the F309S/226aa/N6 variant, and therefore would not encode a FVIII protein, or derivative thereof, having procoagulant activity. Functional, secreted FVIII protein, having procoagulant activity can only be produced after irans-splicing to exon 1 of endogenous human albumin.

Pharmaceutical Compositions

[0109] The pharmaceutical compositions of the present invention contain a

pharmaceutically and/or therapeutically effective amount of at least one nucleic acid construct, lentiviral vector, lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention. In one embodiment of the invention, the effective amount of an agent of the invention per unit dose is an amount sufficient to cause the detectable expression of the gene of interest. In another embodiment of the invention, the effective amount of agent per unit dose is an amount sufficient to prevent, treat or protect against deleterious effects (including severity, duration, or extent of symptoms) of the disease or condition being treated.

[0110] The administration of the pharmaceutical compositions of the invention may be for either "prophylactic" or "therapeutic" purpose. When provided prophylactically, the compositions are provided in advance of any symptom. The prophylactic administration of the composition serves to prevent or ameliorate any subsequent deleterious effects (including severity, duration, or extent of symptoms) of the disease or condition being treated. When provided therapeutically, the composition is provided at (or shortly after) the onset of a symptom of the condition being treated.

[0111] In yet another embodiment of the present invention, for all therapeutic, prophylactic and diagnostic uses, one or more of the aforementioned lentiviral vectors, lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the present invention, as well as other necessary reagents and appropriate devices and accessories, may be provided in kit form so as to be readily available and easily used. Such a kit would comprise a pharmaceutical composition for in vitro or in vivo administration comprising a lentiviral vector of the present invention, and a pharmaceutically acceptable carrier and/or a genetic adjuvant; and instructions for use of the kit.

[0112] The vector may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials. Extemporaneous injection solutions and suspensions may be prepared from purified nucleic acid preparations for the DNA plasmid priming compounds and/or purified viral vector compounds commonly used by one of ordinary skill in the art. Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations may also include other agents commonly used by one of ordinary skill in the art.

[0113] The vector may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, intranasal, intramuscular, subcutaneous, intravenous, intraperitoneal, intraocular, intracranial, intradermal, transdermal (skin patches), topical, or direct injection into a joint or other area of the subject's body. The vector may likewise be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes. An appropriate quantity of LV formulation to be administered is determined by one skilled in the art based on a variety of physical characteristics of the subject or patient, including, for example, the patient's age, body mass index (weight), gender, health, immunocompetence, and the like. Similarly, the volume of administration will vary depending on the route of administration. By way of example, intramuscular injections may range from about 0.1 mL to 1.0 mL. One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired "effective level" in the individual patient.

[0114] The vector of the present invention may be administered through various routes, including, but not limited to, oral, including buccal and sublingual, rectal parenteral, aerosol, nasal, intravenously, subcutaneous, intradermal and topical.

[0115] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. The contents of any patents, patent applications, patent publications, or scientific articles referenced anywhere in this application are herein incorporated in their entirety.

[0116] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. The contents of any patents, patent applications, patent publications, or scientific articles referenced anywhere in this application are herein incorporated in their entirety.

EXAMPLES EXAMPLE 1

[0117] Spliceosome mediated RNA trans-splicing (SMaRT) is one of the few RNA-based technologies that can restrict the production of a protein of therapeutic interest to a specific cell type or organ. In this example, RNA trans-splicing technology was combined with a lentiviral vector (LVs) delivery system to maximize PTM delivery to target cells and achieve persistent gene expression. VSV-G pseudotyped HIV-1 based LVs encoding a PTM were generated that trans-splices human apolipoprotein A-I (hapoA-I) into highly abundant human albumin pre-mRNAs in hepatocytes to increase blood levels of this protein and ultimately correct familial apolipoprotein A-I deficiency. This PTM cassette contains a binding domain targeted to intron 1 of human albumin, splice acceptor sequences and hapoA-I coding sequence. Initial testing of these LV-hapoA-I PTMs were performed in HepG2, and human primary hepatocytes. These cell types produce and process albumin and apoA-I, and therefore are ideal cell models for pre-testing clinical candidate LV-hapoA-I PTMs. The low conservation of intron sequences among species precludes the use of most animal models for pre-testing human PTMs. HepG2 cells and hepatocytes were transduced efficiently by LV- PTMs and analysis of RNA from these cells by RT-PCR for transcripts containing albumin exon 1 fused to hapoA-I coding sequences confirmed accurate trans-splicing into human albumin pre-mRNAs. Levels of hapoA-I PTM RNAs, albumin pre-mRNAs, trans-spliced mRNA, and LV copies per cell were quantified by qRT-PCR and qPCR, respectively; and all four parameters showed clear dose response relationships. Increased MOIs produced a linear increase in the number of LV copies per cell, and cell samples with higher copy values had higher levels of unspliced PTM RNA. Similarly, analysis of trans-splicing level showed a close linear relationship with PTM RNA (Figure) and produced in the range of 5-20 copies of trans-spliced chimeric mRNA per cell in HepG2 and 1-5 copies in human primary hepatocytes. In all samples analyzed a higher level of PTM RNA or target pre-mRNA resulted in higher levels of trans-splicing.

HDL Therapy - SmaRT-Based Strategy

[0118] In the Human Plasma Proteome, the protein breakdown is al -Antitrypsin (3.8%), a2-Macroglobulin (3.6%), Immunoglobulin A (3.4%), Transferrin (3.3%), Hp Type 2-1 (2.9%), IgM (1.98%), Biomarkers (10%), and Albumin (54.3%). This provides the rationale for selecting albumin as a target for trans-splicing. Human Albumin is the most abundant protein in plasma (Human: 35-50 mg/ml; Mouse: 20-30 mg/ml). Albumin is also the most abundant transcript in human liver; human liver produces 12gms/day.

[0119] Apolipoprotein AI is the major component of HDL, plays an important role in promoting the efflux of excess cholesterol to the liver for excretion. The strategy employed in this example is to trans-splice human apolipoprotein A-I (hApoA-I) sequences into highly abundant albumin pre-mRNA target and increase blood levels of human ApoA-I, the major component of high density lipoproteins (HDL) and correct familial apolipoprotein A-I deficiency.

[0120] The trans-splicing into Albumin approach offers several potential advantages over cDNA/recombinant protein therapy. Endogenous regulation - retains endogenous regulation of irans-spliced products, level of irans-splicing is related to level of target pre-mRNA. With SMaRT™ strategy, ApoA-I is produced in hepatocytes and processed in the same cellular compartments as the naturally produced ApoA-I protein. In terms of minimized ectopic expression, irans-splicing occurs only where and when the target pre-mRNA is expressed. Endogenous protein production provides steady ApoA-I levels compared to high-dose / fast elimination of recombinant proteins.

Trans-splicing into Albumin

[0121] The trans-splicing of wild type human apoA-I into highly abundant albumin target pre-mRNA increases expression of human apoA-I protein (FIG. 1). Higher amounts of target pre-mRNA provides a higher irans-splicing efficiency.

Pre-Trans-splicing Molecules (PTMs)

[0122] PTMs are RNAs that are designed to contain a binding domain (BD, -100-150 bp) complementary to a target pre-mRNA, splice elements necessary for irans-splicing and a coding domain, which could be a single exon or entire cDNA (FIG. 2). Target specificity is obtained by tethering the PTM to the target pre-mRNA through base pairing between the BD in the PTM and the target pre-mRNA.

[0123] The LV-hApoA-1 PTM cassette used in these studies employs a Self Inactivating (SIN) configuration, is VSV-G pseudotyped. Employs a liver- specific promoter (HCR- hATT), and contains the payload in a forward orientation (FIG. 3).

Testing LV-hApoA-I PTM in HepG2 & Primary Hepatocytes

[0124] Methods: LV-hApoA-I PTM was tested in HepG2 cells and in human primary hepatocytes. After transduction, HepG2 cells were split and harvested at different days; while, primary hepatocytes were harvested at 72 hrs post-transduction. Total RNA and genomic DNA were analyzed by qRT-PCR and qPCR, respectively (FIG. 4).

PTM Dose Response in HepG2 Cells

[0125] The levels of hApoA-I PTM RNAs, albumin pre-mRNAs, irans-spliced chimeric mRNA, and LV copies per cell are depicted in FIG. 4. All parameters exhibited clear dose response relationships. Increased MOIs produced a linear increase in the number of LV copies per cell leading to higher unspliced PTM RNA. Trans-splicing level showed a close linear relationship with target pre-mRNA and PTM RNA and produced in the range of 5-20 copies of irans-spliced mRNA per cell in HepG2 (FIG. 4).

Trans-splicing in HepG2 cells

[0126] Cells were transduced with VRX1243 and subjected to 8 passages before being plated for transfection. Cells have 6.5 vector (PTM) copies per cell. Human albumin minigene was either cloned in LV backbone (pVRX1200) or pc3.1DNA backbone. Cells were transfected (in duplicate) with equal moles of minigene DNA (LV = 1.7 μg or pc3.1 = 1.5 μg) at -18 hours after plating and cells harvested at 2 days after transfection (FIG. 5).

Trans-Splicing in Human Hepatocytes with VRX1243

[0127] This is the first demonstration of trans-splicing to endogenous albumin in primary human hepatocytes using lentiviral delivery and PTMs. A good dose response was achieved between MOI, and vector, PTM and trans-spliced copies per cell; more vectors per cell produce more PTM and trans-splicing (FIG. 6). Similar dose effects were observed in cell populations with lower target level (data not shown). Target level is not affected at higher transduction levels. Accurate trans-splicing confirmed by sequencing.

Unspliced PTM RNA expression from different MuAlb-HuApaA-1 PTMs

Transduction of VRX1257, 1261 and 1271 in HepG2 cells

[0128] A good dose response (usPTM DNA and proviral copies per cell) was obtained with both VRX1261 and VRX1271. Very high proviral copies per cell at 7 day time for VRX1271 but value is substantially reduced by 14 days; cells transduced at lower dilutions of VRX1271 clearly showed different growth characteristics. A very high unspliced RNA level was obtained per cell; but proviral copies per cell are much higher than expected to achieve in liver. Very good unspliced RNA copies per proviral copy at 14 days (average = 137) were obtained with no major difference in unspliced RNA production per proviral copy between all three vectors (1257, 1261, 1271) (Table 1). Table 1

Summary

[0129] The results in Example 1 demonstrate: i) successful integration of LV delivery platform with SMaRT technology, ii) trans-splicing to a true endogenous pre-mRNA target in human primary hepatocytes, and iii) confirm and extend previous observations of the intimate relationship between PTM RNA, target pre-mRNA and trans-splicing. Trans-splicing was confirmed in various cell lines and human primary hepatocytes with functional promoter(s). A good correlation was obtained between vector dose, pro viral integration, PTM RNA and irans-splicing copies per cell. Experiments are now in progress to assess the therapeutic use of these LV-PTMs in engineered animal models. Results from these studies will serve as a foundation for the future development of liver directed RNA-based therapies.

Claims

CLAIMS What is claimed is:

1. A cell comprising a recombinant lenti viral vector that expresses a nucleic acid molecule encoding a protein or polypeptide of interest wherein said nucleic acid molecule comprises: a) one or more target binding domains that target binding of the nucleic acid molecule that encodes the protein or polypeptide of interest to an abundantly expressed target pre-mRNA within the cell; b) a splice region; c) a spacer region that separates the splice region from the target binding domain; and d) a nucleotide sequence encoding the a protein or polypeptide of interest to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

2. The cell of claim 1 wherein the splice region comprises either i) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' splice acceptor site; or ii) a 5' splice site or 5' donor site, or a combination of both.

3. The cell of claim 1, wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3' splice region.

4. The cell of claim 1 wherein the abundantly expressed target pre-mRNA is selected from the group consisting of pre-mRNAs encoding albumin, casein, myosin and fibroin.

5. The cell of claim 1 wherein the abundantly expressed target pre-mRNA encodes albumin.

6. The cell of claim 1 wherein the abundantly expressed target pre-mRNA encodes casein.

7. The cell of claim 1 wherein the abundantly expressed target pre-mRNA comprises a tumor-specific or tumor associated transcript.

8. The cell of claim 1 wherein the protein or polypeptide of interest is selected from the group consisting of cytokines, growth factors, insulin, hormones, enzymes and antibody polypeptides.

9. The cell of claim 1 wherein the protein or polypeptide of interest is selected from the group consisting of ApoAl, a pre-pro-ApoAl, ApoAl milano variant, and ApoB.

10. The cell of claim 1 wherein the protein or polypeptide of interest comprises a single chain antibody polypeptide.

11. The cell of claim 1 wherein the protein or polypeptide of interest comprises Factor VIII protein.

12. A recombinant lentiviral vector that expresses a nucleic acid molecule encoding a protein or polypeptide of interest wherein said nucleic acid molecule comprises: a) one or more target binding domains that target binding of the nucleic acid molecule that encodes the protein or polypeptide of interest to an abundantly expressed target pre-mRNA within the cell; b) a splice region; c) a spacer region that separates the splice region from the target binding domain; and d) a nucleotide sequence encoding the protein or polypeptide of interest to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

13. The recombinant lentiviral vector of claim 12 wherein the splice region comprises either i) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' splice acceptor site; or ii) a 5' splice site or 5' donor site, or a combination of both.

14. The recombinant lentiviral vector of claim 12, wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3' splice region.

15. The recombinant lentiviral vector of claim 12 wherein the abundantly expressed target pre-mRNA is selected from the group consisting of pre-mRNAs encoding albumin, casein, myosin and fibroin.

16. The recombinant lentiviral vector of claim 12 wherein the abundantly expressed target pre-mRNA encodes albumin.

17. The recombinant lentiviral vector of claim 12 wherein the abundantly expressed target pre-mRNA encodes casein.

18. The recombinant lentiviral vector of claim 12 wherein the abundantly expressed target pre-mRNA comprises a tumor-specific or tumor associated transcript.

19. The recombinant lentiviral vector of claim 12 wherein the protein or polypeptide of interest is selected from the group consisting of cytokines, growth factors, insulin, hormones, enzymes and antibody polypeptides.

20. The recombinant lentiviral vector of claim 12 wherein the protein or polypeptide of interest is selected from the group consisting of ApoAl, a pre-pro-ApoAl, ApoAl milano variant, and ApoB.

21. The recombinant lentiviral of claim 12 wherein the protein or polypeptide of interest comprises a single chain antibody polypeptide.

22. The recombinant lentiviral of claim 12 wherein the protein or polypeptide of interest comprises Factor VIII protein.

23. A method of producing a chimeric RNA molecule that encodes a protein or polypeptide of interest in a cell comprising: contacting an abundantly expressed target pre- mRNA within the cell with a nucleic acid molecule expressed by a recombinant lentiviral vector wherein the nucleic acid molecule encoding the protein or polypeptide of interest that is recognized by nuclear splicing components wherein said nucleic acid molecule comprises: a) one or more target binding domains that target binding of the nucleic acid molecule that encodes the protein or polypeptide of interest to the abundantly expressed target pre-mRNA within the cell; b) a splice region; c) a spacer region that separates the splice region from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre- mRNA; wherein the nucleic acid molecule is recognized by nuclear splicing components within the cell.

24. The method of claim 23 wherein the abundantly expressed target pre-mRNA is selected from the group consisting of pre-mRNAs encoding albumin, casein, myosin and fibroin.

25. The method of claim 23 wherein the abundantly expressed target pre-mRNA encodes albumin.

26. The method of claim 23 wherein the abundantly expressed target pre-mRNA encodes casein.

27. The method of claim 23 wherein the abundantly expressed target pre-mRNA comprises a tumor-specific or tumor associated transcript.

28. The method of claim 23 wherein the protein or polypeptide of interest is selected from the group consisting of cytokines, growth factors, insulin, hormones, enzymes and antibody polypeptides.

29. The method of claim 23 wherein the protein or polypeptide of interest is selected from the group consisting of ApoAl, a pre-pro-ApoAl, ApoAl milano variant, and ApoB.

30. The method of claim 23 wherein the protein or polypeptide of interest comprises a single chain antibody polypeptide.

31. The method of claim 23 wherein the protein or polypeptide of interest comprises Factor VIII protein.

32. A method of producing a protein or polypeptide of interest in a cell comprising: contacting an abundantly expressed target pre-mRNA within the cell with a nucleic acid molecule expressed by a recombinant lentiviral vector so as to produce a chimeric RNA molecule that encodes the protein or polypeptide of interest wherein said nucleic acid molecule comprises: a) one or more target binding domains that target binding of the nucleic acid molecule to the abundantly expressed target pre-mRNA within the cell; b) a splice region; c) a spacer region that separates the splice region from the target binding domain; and d) a nucleotide sequence to be trans-spliced to the target pre-mRNA; wherein the nucleic acid molecule is recognized by nuclear splicing components within the cell and wherein the chimeric RNA molecule is translated by the cell to produce the protein or polypeptide of interest.

33. The method of claim 32 wherein the abundantly expressed target pre-mRNA is selected from the group consisting of pre-mRNAs encoding albumin, casein, myosin and fibroin.

34. The method of claim 32 wherein the abundantly expressed target pre-mRNA encodes albumin.

35. The method of claim 32 wherein the abundantly expressed target pre-mRNA encodes casein.

36. The method of claim 32 wherein the abundantly expressed target pre-mRNA comprises a tumor-specific or tumor associated transcript.

37. The method of claim 32 wherein the protein or polypeptide of interest is selected from the group consisting of cytokines, growth factors, insulin, hormones, enzymes and antibody polypeptides.

38. The method of claim 32 wherein the protein or polypeptide of interest is selected from the group consisting of ApoAl, a pre-pro-ApoAl, ApoAl milano variant, and ApoB.

39. The method of claim 32 wherein the protein or polypeptide of interest comprises a single chain antibody polypeptide.

40. The method of claim 32 wherein the protein or polypeptide of interest comprises Factor VIII protein.

41. The recombinant lentiviral vector of claim 12 wherein the target pre-mRNA is expressed within a liver cell.

42. The recombinant lentiviral vector of claim 12 wherein the liver cell is a parenchymal cell such as a hepatocyte, or a non-parenchymal cell such as an endothelial cell, a Kupffer cell, a stellate cell, oval cell, or any of the precursors thereto such as hepatic stem cells, bone marrow liver stem cells.

43. The recombinant vector of claim 12 wherein expression of the nucleic acid molecule is controlled by a liver cell specific promoter.