WO2012159120A2

WO2012159120A2 - Gene therapy based strategy for treating hiv

Info

Publication number: WO2012159120A2
Application number: PCT/US2012/038901
Authority: WO
Inventors: Lung-Ji Chang
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2011-05-19
Filing date: 2012-05-21
Publication date: 2012-11-22
Also published as: WO2012159120A3

Abstract

Disclosed herein are methods and compositions for treating or increasing resistance HIV infection. Vectors carrying a codon-optimized CCR5delta32 gene, Hl-promoter driven CCR5shRNA and/or a triple miRNA (microRNA) intronic cassette (miR155, 19a, 30a) against HIV-1 pol, int and vpu may be packaged into virus particles. In a specific embodiment, vectors carrying the CCR5shRNA and the triple miRNA against HIV-1, pol, int and vpu without the CCR5delta32 gene are provided. Significant resistance to HIV-1 infection and envelope mediated fusion was observed in the vector-modified HOS-R5 cells that endogenously express CD4, CCR5 and CXCR4. Expression of endogenous CCR5 was inhibited more than 90% after vector CCR5shRNA gene transfer as demonstrated by flow cytometry.

Description

GENE THERAPY BASED STRATEGY FOR TREATING HIV

Cross-reference to related applications

This application is related to U.S. Provisional Application No. 61/487,726 filed on May 19, 2011 to which priority is claimed under 35 USC 119.

Background

HIV-1 infection is associated with viral persistence, rapid virus spread and CD4 T cell turnover that systematically damages host immune functions.¹ Treatment of HIV infection by antiretroviral therapy is effective but costly and often associated with numerous side effects. This highlights the need for a one-time and durable treatment against this deadly disease. The key to a permanent treatment to chronic HIV infections is to elicit potent host resistance to viral infection and to restore immune functions. The prolonged incubation period of HIV-1 provides a good opportunity for applying non-conventional interventions such as gene therapy. For HIV gene therapy to be effective, the combination of an efficient gene transfer vector and a powerful anti-HIV strategy is necessary.

Summary

Embodiments of the present invention pertain to an innovative gene therapy strategy that offers an effective treatment to infected individuals. HIV resistance is established in patients' HSCs by lentiviral transduction of (i) a small hairpin RNA (shRNA) to block endogenous CCR5 expression, (ii) a sequence-modified CCR5A32 gene to interfere with the function of native CCR5 and CXCR4 and/or (iii) effective anti-HIV shRNA genes to target viral RNAs. The CCR5A32 will block viral entry and the shRNAs can suppress HIV replication. For gene therapy to be effective in HIV patients, efficient delivery and long term stability are necessary. The materials and methods for a state-of-the-art lentiviral vector (LV) system, the effective shRNAs against HIV-1, and the mechanism of CCR5A32 repressing both CCR5 and CXCR4 have been well-established in our laboratories.

An advanced gene and cell based therapeutic approach against HIV/ AIDS is developed using lentiviral vectors (LVs) encoding (1) shRNAs targeting endogenous CCR5, and (2) shRNAs targeting highly conserved HIV-1 sequences and, optionally, (3) CCR5delta32 that can down-regulate endogenous CCR5 and CXCR4.

LVs carrying a codon-optimized CCR5delta32 gene, HI -promoter driven CCR5shRNA and/or a triple miRNA (microRNA) intronic cassette (miR155, 19a, 30a) against HIV-1 pol, int and vpu were packaged into virus particles. In a specific embodiment, LVs carrying the CCR5shRNA and the triple miRNA against HIV-1, pol, int and vpu without the CCR5delta32 gene are provided.

Significant resistance to HIV-1 infection and envelope mediated fusion was observed in the LV-modified HOS-R5 cells that endogenously express CD4, CCR5 and CXCR4. Expression of endogenous CCR5 was inhibited more than 90% after LV CCR5shRNA gene transfer as demonstrated by flow cytometry.

LV transduction of CCR5 shRNA, codon-optimized CCR5delta32 and the triple miRNA intronic cassettes, individually or in combination, demonstrated various degrees of antiviral effects. Resistance to HIV persisted after prolonged passages of the LV-transduced HOS-R5 cells, demonstrating the stability of the integrated anti- HIV provirus. According to another embodiment, the invention pertains to a population of genetically modified stem cells that can establish stable HIV resistance in all lineages of differentiated cells after autologous cell transplantation. For example, genetically modified stem cells expressing CCR5 shRNA and the triple miRNA cassettes, optionally with the CCR5delta32 gene are provided. It has been found that the CCR5delta32 gene can cause cytotoxicity in some instances, and therefore, certain embodiments are specifically designed to avoid the inclusion of the CCR5delta 32 gene.

Brief Description of Drawings

Fig. 1. An improved LV system for the expression of

and multiple shRNAs.

Fig. 2. Inhibition of primary HIV-1 isolates by LVshRNAs. PBMCs were activated by anti-CD3/CD28 Abs, transduced with LVshRNAs and challenged with HIV-1. HIV- 1 p24 ELIS A kinetics were determined as shown.

Fig. 3. Effect of

and CCR5shRNA on CCR5 and CXCR4 expression. HOS-R5 or HOS-X4 cells were infected with lentivectors as indicated. Note that HOS-R5 cells express both CCR5 and CXCR4.

Fig. 3'. CD4 T cells transduced with

display HIV-1 resistance.

Fig. 4. R5-HIV-1 (NL-AD8) challenge of LV-transduced CCR5-Magi cells. (A) HIV-1 infectivity assay by LTR-lacZ reporter cells. Blue cells are infected cells. (B) HIV-1 syncytium formation assay.

Detailed Description

According to one embodiment, the invention pertains to a composition that includes one or more genetic components. The one or more genetic components include (a) at least one shRNA or miRNA targeting endogenous CCR5; (b) at least one miRNA targeting at least one of pol, int, or vpui; and/or (c) a CCR5delta32 gene (see Appendix A). In a more specific embodiment, elements (a), (b) and (c) are all packaged into a vector. In an alternative embodiment, only elements (a) and (b) are provided. In a typical embodiment, the vector is a viral vector suitable for human administration. One specific example of a viral vector includes lentiviral viral vectors. Element (b) may include a first, a second and a third miRNA, wherein the first, second and third miRNA target pol, int, and vpu, respectively. In another specific embodiment, the CCR5delta32 gene is codon optimized. In an alternative embodiment, the CCR5delta32 gene is not codon optimized. The composition may further include a pharmaceutically acceptable carrier.

According to another embodiment, the invention pertains to a viral vector engineered to include a codon-optimized or non-optimized CCR5delta32gene; an HI promoter driven CCR5shRNA or a CCR5 miRNA; and/or a triple miRNA intronic cassette against HIV-1 pol, int and/or vpu.

Another embodiment of the invention pertains to a method of treating a subject who has contracted HIV. In a specific embodiment, the method includes administering a therapeutically effective amount of a composition that includes (a) at least one shRNA or miRNA targeting endogenous CCR5; (b) at least one miRNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene. In an alternative embodiment, the method involves administering elements (a) and (b) without (c).

According to a further embodiment, the invention pertains to a composition that includes a population of cells that have been transduced with (a) at least one shRNA or miRNA targeting endogenous CCR5; (b) at least one miRNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene. The population of cells may be transduced in vitro obtained from a subject. Typically, the cells are transduced with a viral vector comprising a codon-optimized (or alternatively, non- optimized or "native") CCR5delta32gene; an HI promoter driven CCR5shRNA or CCR5miRNA; and a triple miRNA intronic cassette against HIV-1 pol, int and vpu. Alternatively, the population of cells may be transduced with a viral vector comprising an HI promoter driven CCR5shRNA or CCR5miRNA; and a triple miRNA intronic cassette against HIV- 1 pol, int and vpu.

In yet another embodiment, the invention pertains to a method of treating a subject who has contracted or who has been exposed to HIV. The method includes administering a therapeutically effective amount of cells that have been transduced with (a) at least one shRNA or miRNA targeting endogenous CCR5; (b) at least one miRNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene to the subject. In a typical embodiment, the cells are autologous to the subject. In a specific embodiment, the cells are stem cells. In an even more specific embodiment, the cells are hematopoietic stem cells.

According to another embodiment, provided is a method of inducing resistance to HIV comprising administering to a subject cells that have been transformed to express (a) at least one shRNA or miRNA targeting endogenous CCR5; (b) at least one miRNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene. Alternatively, examples include viral vectors modified to express (a) and (b) without (c).

As used herein, the terms "express" or "expression" in relation to genes or nucleic acid sequences, is meant to refer to the production of the corresponding gene product. Typically, gene products are proteins encoding by genes, but includes the production functional RNA products. In accordance with the method of the invention, an expression vector is a viral or a non-viral expression vector. Viral expression vectors which may be used advantageously in the method of the invention include, but are not limited to, an adeno associated virus (AAV) vector, a lentivirus vector, an adenovirus vector, and a herpes simplex virus (HSV) vector.

Viral Vectors

In one embodiment, compositions are provided which are tailored to include a nucleic acid sequence encoding certain genetic components (e.g., (a) at least one shRNA or miRNA targeting endogenous CCR5; (b) at least one miRNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene; alternatively, (a) and (b) without (c)) in an expression vector. Viral vectors for use in the invention are those that exhibit low toxicity to a host cell. Viral vector methods and protocols that may be used in the invention are reviewed in Kay et al. Nature Medicine 7:33-40, 2001. The use of specific vectors, including those based on adenoviruses, adeno-associated viruses, herpes viruses, and retroviruses are described in more detail below.

The use of recombinant adenoviruses as vectors is discussed in W. C. Russell, Journal of General Virology 81:2573-2604, 2000; and Bramson et al., Curr. Opin. Biotechnol. 6:590-595, 1995. Adenovirus vectors are preferred for use in the invention because they (1) are capable of highly efficient gene expression in target cells and (2) can accommodate a relatively large amount of heterologous (non- viral) DNA. A preferred form of recombinant adenovirus is a "gutless", "high-capacity", or "helper-dependent" adenovirus vector. Such a vector features, for example, (1) the deletion of all or most viral-coding sequences (those sequences encoding viral proteins), (2) the viral inverted terminal repeats (ITRs) which are sequences required for viral DNA replication, (3) up to 28-32 kb of "exogenous" or "heterologous" sequences (e.g., sequences encoding an ammonia producing enzyme), and (4) the viral DNA packaging sequence which is required for packaging of the viral genomes into infectious capsids.

Other viral vectors that might be used in the invention are adeno-associated virus (AAV)-based vectors. AAV-based vectors are advantageous because they exhibit high transduction efficiency of target cells and can integrate into the host genome in a site-specific manner. Use of recombinant AAV vectors is discussed in detail in Tal, J., J. Biomed. Sci. 7:279-291, 2000 and Monahan and Samulski, Gene Therapy 7:24-30, 2000. A typical AAV vector comprises a pair of AAV inverted terminal repeats which flank at least one cassette containing a tissue (e.g., gum)- or cell-specific promoter operably linked to a urease nucleic acid. The DNA sequence of the AAV vector, including the LTRs, the promoter and, for example, urease gene may be integrated into the host genome.

The use of herpes simplex virus (HSV)-based vectors is discussed in detail in Cotter and Robertson, Curr. Opin. Mol. Ther. 1 :633-644, 1999. HSV vectors deleted of one or more immediate early genes (IE) are advantageous because they are generally non-cytotoxic, persist in a state similar to latency in the host cell, and afford efficient host cell transduction. Recombinant HSV vectors can incorporate approximately 30 kb of heterologous nucleic acid. A preferred HSV vector is one that: (1) is engineered from HSV type I, (2) has its IE genes deleted, and (3) contains a tissue-specific promoter operably linked to a urease nucleic acid. HSV amplicon vectors may also be useful in various methods of the invention. Typically, HSV amplicon vectors are approximately 15 kb in length, and possess a viral origin of replication and packaging sequences. Retroviruses such as C-type retroviruses and lentiviruses might also be used in the invention. For example, retroviral vectors may be based on murine leukemia virus (MLV). See, e.g., Hu and Pathak, Pharmacol. Rev. 52:493-511, 2000 and Fong et al., Crit. Rev. Ther. Drug Carrier Syst. 17:1-60, 2000. MLV-based vectors may contain up to 8 kb of heterologous (therapeutic) DNA in place of the viral genes.

Additional retroviral vectors that might be used are replication-defective lentivirus-based vectors, including human immunodeficiency (HlV)-based vectors. See, e.g., Vigna and Naldini, J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol. 72:8150-8157, 1998. Lentiviral vectors are advantageous in that they are capable of infecting both actively dividing and non-dividing cells. They are also highly efficient at transducing human epithelial cells. Lentiviral vectors for use in the invention may be derived from human and non-human (including SW) lentiviruses.

A lentiviral vector may be packaged into any suitable lentiviral capsid. The substitution of one particle protein with another from a different virus is referred to as "pseudotyping". The vector capsid may contain viral envelope proteins from other viruses, including murine leukemia virus (MLV) or vesicular stomatitis virus (VSV). The use of the VSV G-protein yields a high vector titer and results in greater stability of the vector virus particles.

Alphavirus-based vectors, such as those made from semliki forest virus (SFV) and sindbis virus (SIN), might also be used in the invention. Use of alphaviruses is described in Lundstrom, K., Intervirology 43:247-257, 2000 and Perri et al., Journal of Virology 74:9802-9807, 2000. Alphavirus vectors typically are constructed in a format known as a replicon. A replicon may contain (1) alphavirus genetic elements required for RNA replication, and (2) a nucleic acid encoding the (a), (b) and/or (c) elements discussed above. Within an alphavirus replicon, the heterologous nucleic acid may be operably linked to a tissue-specific promoter or enhancer.

Recombinant, replication-defective alphavirus vectors are advantageous because they are capable of high-level heterologous (therapeutic) gene expression, and can infect a wide host cell range. Alphavirus replicons may be targeted to specific cell types by displaying on their virion surface a functional heterologous ligand or binding domain that would allow selective binding to target cells expressing a cognate binding partner. Alphavirus replicons may establish latency, and therefore long-term heterologous nucleic acid expression in a host cell. The replicons may also exhibit transient heterologous nucleic acid expression in the host cell. A preferred alphavirus vector or replicon is non-cytopathic.

In many of the viral vectors compatible with methods of the invention, more than one promoter can be included in the vector to allow more than one heterologous gene to be expressed by the vector. Further, the vector can comprise a sequence which encodes a signal peptide or other moiety which facilitates the secretion of a gene product from the host cell.

To combine advantageous properties of two viral vector systems, hybrid viral vectors may be used to deliver a nucleic acid to a target tissue. Standard techniques for the construction of hybrid vectors are well-known to those skilled in the art. Such techniques can be found, for example, in Sambrook, et al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor, N. Y. or any number of laboratory manuals that discuss recombinant DNA technology. Double- stranded AAV genomes in adenoviral capsids containing a combination of AAV and adenoviral lilts may be used to transduce cells. In another variation, an AAV vector may be placed into a

"gutless", "helper-dependent" or "high-capacity" adenoviral vector. Adenovirus/ AAV hybrid vectors are discussed in Lieber et al., J. Virol. 73:9314-9324, 1999.

Retrovirus/adenovirus hybrid vectors are discussed in Zheng et al., Nature Biotechnol. 18:176-186, 2000. Retroviral genomes contained within an adenovirus may integrate within the host cell genome and effect stable urease gene expression.

Other nucleotide sequence elements which facilitate expression of (a) at least one shRNA or miRNA targeting endogenous CCR5 ; (b) at least one miRNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene; or (a) and (b) without (c) and cloning of the vector are further contemplated. For example, the presence of enhancers upstream of the promoter or terminators downstream of the coding region, for example, can facilitate expression.

Other Delivery Vehicles

Many nonviral techniques for the delivery of a nucleic acid sequence into a cell can be used, including direct naked DNA uptake (e.g., Wolff et al., Science 247: 1465-1468, 1990), receptor-mediated DNA uptake, e.g., using DNA coupled to asialoorosomucoid which is taken up by the asialoglycoprotein receptor in the liver (Wu and Wu, J. Biol. Chem. 262: 4429-4432, 1987; Wu et al., J. Biol. Chem. 266: 14338-14342, 1991), and liposome-mediated delivery (e.g., Kaneda et al., Expt. Cell Res. 173: 56-69, 1987; Kaneda et al., Science 243: 375-378, 1989; Zhu et al., Science 261: 209-211, 1993). Many of these physical methods can be combined with one another and with viral techniques; enhancement of receptor-mediated DNA uptake can be effected, for example, by combining its use with adenovirus (Curiel et al., Proc. Natl. Acad. Sci. USA 88: 8850-8854, 1991 ; Cristiano et al., Proc. Natl. Acad. Sci. USA 90: 2122-2126, 1993). Other examples include stem cells such as mesenchymal stem cells, hematopoietic stem cells, cardiac stem cells or neural stem cells, embryonic stem cells that have been engineered to express (a) at least one shRNA or miRNA targeting endogenous CCR5; (b) at least one miRNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene; or to express (a) and (b) without (c). Such stem cells can be administered in such a way to be incorporated in to tissues of a patient in need. In a particular embodiment, stem cells are administered to myocardial tissue. Bu L, et al "Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages" Nature 2009 460:113-117.

A subject as used herein is a human or other primate. Typically, a subject is one who has been diagnosed with HIV, who is exhibiting one or more symptoms of HIV, who has been exposed to HIV, or who is engages in activity known to have a high risk of HIV transmission (e.g. medical personnel).

RNA interference (RNAi) is a process by which double- stranded RNA (dsRNA) is used to silence gene expression. While not wanting to be bound by theory, RNAi begins with the cleavage of longer dsRNAs into small interfering RNAs (siRNAs) by an RNaselll-like enzyme, dicer. SiRNAs are dsRNAs that are usually about 19 to 28 nucleotides, or 20 to 25 nucleotides, or 21 to 22 nucleotides in length and often contain 2-nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini. One strand of the siRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). RISC uses this siRNA strand to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then cleaves these target mRNAs or inhibits their translation. Therefore, the siRNA strand that is incorporated into RISC is known as the guide strand or the antisense strand. The other siRNA strand, known as the passenger strand or the sense strand, is eliminated from the siRNA and is at least partially homologous to the target mRNA. Those of skill in the art will recognize that, in principle, either strand of an siRNA can be incorporated into RISC and function as a guide strand. However, siRNA design (e.g., decreased siRNA duplex stability at the 5' end of the desired guide strand) can favor incorporation of the desired guide strand into RISC.

The antisense strand of an siRNA is the active guiding agent of the siRNA in that the antisense strand is incorporated into RISC, thus allowing RISC to identify target mRNAs with at least partial complementarity to the antisense siRNA strand for cleavage or translational repression. RISC-related cleavage of mRNAs having a sequence at least partially complementary to the guide strand leads to a decrease in the steady state level of that mRNA and of the corresponding protein encoded by this mRNA. Alternatively, RISC can also decrease expression of the corresponding protein via translational repression without cleavage of the target mRNA.

The term "siRNA" as used herein refers to a double- stranded interfering RNA unless otherwise noted. Typically, an siRNA of the invention is a double- stranded nucleic acid molecule comprising two nucleotide strands, each strand having about 19 to about 28 nucleotides (i.e. about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). The phrase "interfering RNA having a length of 19 to 49 nucleotides" when referring to a double- stranded interfering RNA means that the antisense and sense strands independently have a length of about 19 to about 49 nucleotides, including interfering RNA molecules where the sense and antisense strands are connected by a linker molecule.

In addition to siRNA molecules, other interfering RNA molecules and RNA- like molecules can interact with RISC and silence gene expression. Examples of other interfering RNA molecules that can interact with RISC include short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs (miRNAs), and dicer- substrate 27- mer duplexes. Examples of RNA-like molecules that can interact with RISC include siRNA, single- stranded siRNA, microRNA, and shRNA molecules containing one or more chemically modified nucleotides, one or more non-nucleotides, one or more deoxyribonucleo tides, and/or one or more non-phosphodiester linkages. All RNA or RNA-like molecules that can interact with RISC and participate in RISC-related changes in gene expression are referred to herein as "interfering RNAs" or

"interfering RNA molecules." SiRNAs, single- stranded siRNAs, shRNAs, miRNAs, and dicer- substrate 27-mer duplexes are, therefore, subsets of "interfering RNAs" or "interfering RNA molecules."

Single- stranded interfering RNA has been found to effect mRNA silencing, albeit less efficiently than double- stranded RNA. Therefore, embodiments of the present invention also provide for administration of a single-stranded interfering RNA that has a region of at least near-perfect contiguous complementarity with a portion of the CCR5 RNA, or vpu, int, or pol of HIV. The single-stranded interfering RNA has a length of about 19 to about 49 nucleotides as for the double-stranded interfering RNA cited above. The single- stranded interfering RNA has a 5' phosphate or is phosphorylated in situ or in vivo at the 5' position. The term "5' phosphorylated" is used to describe, for example, polynucleotides or oligonucleotides having a phosphate group attached via ester linkage to the C5 hydroxyl of the sugar (e.g., ribose, deoxyribose, or an analog of same) at the 5' end of the polynucleotide or

oligonucleotide.

Single-stranded interfering RNAs can be synthesized chemically or by in vitro transcription or expressed endogenously from vectors or expression cassettes as described herein in reference to double- stranded interfering RNAs. 5' Phosphate groups may be added via a kinase, or a 5' phosphate may be the result of nuclease cleavage of an RNA. A hairpin interfering RNA is a single molecule (e.g., a single oligonucleotide chain) that comprises both the sense and antisense strands of an interfering RNA in a stem-loop or hairpin structure (e.g., a shRNA). For example, shRNAs can be expressed from DNA vectors in which the DNA oligonucleotides encoding a sense interfering RNA strand are linked to the DNA oligonucleotides encoding the reverse complementary antisense interfering RNA strand by a short spacer. If needed for the chosen expression vector, 3' terminal T's and nucleotides forming restriction sites may be added. The resulting RNA transcript folds back onto itself to form a stem-loop structure.

Examples

Example 1: A safe and efficient lentiviral vector system. An advanced HIV-1- derived LV system (NHP/TYF) has been developed that infects both dividing and non-dividing cells and integrate into target cells with high efficiency allowing permanent expression of therapeutic gene(s).³' ⁴ The NHP/TYF LV system is derived from HIV-1_NL4-₃ with extensive deletions of viral sequences; the NHP DNA encodes viral gag-pol, tat and rev with deletions of LTR, 5' leader, most of env and all of the 3' HIV elements; the TYF DNA synthesizes the vector genome that carries minimal LTR sequences (self-inactivating), part of gag, part of env including RRE, polypurine tracts and a critical mutation in the packaging signal that deletes the major 5' splice site (Fig.l). The TYF vector contains a pol III HI promoter for the expression of an siRNA in the opposite orientation and a pol II promoter for the expression of CCR5A32 and three introns for the synthesis of three microRNAs.

When co-transfected with a VSV-G envelop plasmid, lentiviral particles are produced that can efficiently infect many different cell types including HSCs. The vector production protocol has been streamlined to produce >10¹⁰ infectious units/ml of vectors. The extensive safety profile of the NHP/TYF LV system has been established based on viral p24 ELISA, PCR and prolonged co-culture of the NHP/TYF/VSV-G cotransfected cells with an HIV-1 -susceptible human lymphoma cell line, and no replication-competent virus was detected.³

Example 2: Anti-HIV shRNAs targeting highly conserved HIV-1 sequences. The success of treating HIV infection by shRNAs depends upon the high efficiency of inhibition of viral replication to prevent possible escape mutants. Unless the shRNA targets a highly conserved HIV sequence, escape mutants are certain to arise.⁵ Although some criteria have been reported that can help identify effective shRNA target sites, the best target sites in the HIV-1 genome still need to be determined empirically.²' ⁶ A rational approach of anti-HIV LV shRNA gene therapy is to target highly conserved regions in the viral genome. Accordingly, we have identified several highly conserved shRNA target sites after screening multiple regions across the entire HIV-1 genome.⁷ We have further modified four of the HIV-1 shRNA target sites: poli, inti, vpui and revi with extension of the target sequence from 21nt to 28 nt. These LVshRNAs were tested using peripheral blood mononuclear cells (PBMCs) challenged by several primary HIV-1 isolates and demonstrated an average of 60-90% virus inhibition effects based on HIV-1 p24 ELISA kinetics (representative 4- week viral kinetics shown in Fig. 2). It was found that the most consistent anti-HIV shRNAs are the poli and the inti targeting HIV pol and int, respectively.

Example 3: Dominant negative effect of CCR5A32 down-regulates both CCR5 and CXCR4. Since the discovery of CCR5 as a key coreceptor for HIV entry, the protective phenotype of CCR5A32 has gradually been revealed and the first CCR5- based entry inhibitor has been recently approved by the FDA.⁸' ⁹ It is now evident that stable expression of the naturally occurring CCR5A32 protein actively confers resistance to HIV-1, as the lack of protection of CCR5(-/-) individuals correlates well with the loss of expression of CCR5A32.¹⁰ The suppression of CXCR4 expression by CCR5A32 and suppression of CCR5 by CCR5shRNA are demonstrated in Fig. 3.

Example 4: In vitro culture systems for T cell and dendritic cell (DC) development. To examine the effect of LVs on adult human immune cells, a convenient model is needed. The RAG2-/-YC-/- mouse-human immune reconstitution model has demonstrated the development of all lineages of immune cells using human cord blood HSCs;¹² however, HSCs from adult donors are not compatible with this model. To generate mature T cells from adult HSCs, we have established a convenient in vitro model based on mouse stromal cells expressing Delta-like 1 (OP9- DL1), Flt3 ligand and interleukin-7 (IL-7).¹³ Adult human CD34+ HSCs cultured under this condition result in robust expansion of preT cells which develop into mature CD4 T cells.¹⁴ This convenient in vitro T cell development system is useful for the analysis of LV effects on HSC development and T cell functions. The developing pre-T and pro-T cells can be analyzed at various time points along the T cell development pathway. Molecular analyses of the HSCs and the derived T cells could offer further insight into the long term effects of LVs on the development of HSCs to T cells.

In addition to T cells, DCs also play an important role in both innate and adaptive immunity. There are multiple lineages of DCs in the hematopoietic system,¹⁵ and the development process of DCs is not well characterized. Using a series of LV-engineered stromal cell lines expressing multiple growth factors, we have established a DC development culture system from adult human HSCs (manuscript submitted). DC progenitors were generated and expanded in the stromal culture in 20-30 days and the resulted mature DCs displayed morphology, phenotype and T cell stimulation functions similar to that of the monocyte-derived DCs. These novel human adult HSC-to-T cell and HSC-to-DC development systems are unique preclinical evaluation tools available in hands for long-term safety and efficacy evaluation of the proposed gene therapy strategy.

Example 5: Construction of LVs expressin2 codon-optimized, pol II promoter- driven CCR5A32, and pol II/pol III promoter-driven shRNAs inhibiting the native CCR5 and HIV-1 (LV-R5A32mirHIV). LVs were constructed to express a codon-optimized CCR5A32 gene under control of the pol II EFlot promoter. A microRNA targeting the endogenous CCR5 was cloned into the intron of the LV that contains three introns (as shown in Fig. 1 and Appendix A). Two additional microRNAs based on the backbone of mir30a and mirl55, targeting highly conserved sequences in the HIV- 1 genome, poli and inti, which have demonstrated efficient anti- HIV effects (as shown in Fig. 2), were also cloned into the same vector. An additional HIV-specific shRNA, vpui, was cloned behind an HI pol III promoter in the opposite orientation in the vector. LVs encoding three microRNAs in the three introns have been generated in the past and shown to effectively suppress three different RNA targets.

The expression of CCR5A32 and the suppression of endogenous CCR5 and CXCR4 is examined in activated PBMCs by Western blotting and flow cytometry using anti-CCR5 and anti-CXCR4 antibodies. To see if there is a synergistic effect from the combination of multiple shRNAs targeting HIV-1, lenti viral constructs carrying one are compared versus multiple shRNAs.⁷

Example 6: Analysis of the function of CCR5A32 and suppression of endogenous CCR5 in human lymphocytes transduced with LV-R5A32shHIV. The CCR5- specific microRNA represses the expression of the endogenous CCR5 by targeting the cognate messenger RNAs for degradation or translational repression. The ectopic expression of the CCR5A32 suprresses the expression of the native CCR5 and CXCR4 (Fig. 3). The effect of CCR5A32 in the presence or absence of the CCR5- specific microRNA is examined using a CCR5+CXCR4+CD4+ T cell reporter cell line. After LV-R5A32shHIV transduction, the expression of CCR5 and CXCR4 is analyzed in the T cells by Western blotting and flow cytometry. LV-R5A32shHIV gene transfer will increase the expression of CCR5A32, which will suppress both CCR5 and CXCR4. The CCR5-specific microRNA targeting endogenous CCR5 will further down-regulate the expression of CCR5.

Example 7: Demonstration of inhibition of HIV-1 replication by LV- R5A32shHIV in CD4 T cells and PBMCs. The effects of CCR5A32 and shRNAs targeting CCR5 and HIV-1 will be examined using CD4 T cell lines and PBMCs using established methods. The LV-transduced CD4 T cells and activated PBMCs will be challenged with HIV-1 carrying a reporter gfp gene (HIVgfp) and viral replication will be monitored by analysis of the kinetics of GFP and p24. CD4 T cells and PBMCs will become resistant to HIV-1 infection after transduction by LV- R5A32shHIV but not by a control LV-shRNA. Example 8: Transduction of HSCs by LV-R5A32shHIV and development of mature CD4 T cells. We have demonstrated efficient in vitro development of mature CD4 T cells from adult CD34+ HSCs using an improved OP9 stromal cell culture system. This is accomplished by timely removal of IL-7 during the transition of proT cells from double negative to double positive stages (manuscript submitted). Human adult HSCs are isolated and transduced by LVs and cultured under the in vitro T cell development condition on the OP9 stromal cells as described.¹⁴ The LV-modified HSCs are compared to control HSCs for the analyses of kinetics and functions of T cell development. The effect of LV transduction on human adult HSC development into mature T cells is determined.

Example 9: Analysis of functional DC development from HSCs after LV- R5A32shHIV gene transfer. In addition to T cell development, the possible effects of lentiviral gene transfer on DC development are investigated using a novel HSC to DC development culture system established in the Pi's laboratory. Adult human HSCs are cultured on OP9-GMCSF-IL-15 feeder cells and functional DCs are developed in 20-30 days with increased expression of DC markers (published). Control LV and LV-R5A32shHIV transduced CD34+ HSCs are compared and the expression kinetics of transcriptional factors specific to DC development is characterized, and the antigen capture and T cell activation functions of the in vitro developed DCs is characterized. Little to no adverse effect on DC development due to the lentiviral gene transfer is achieved.

Example 10: Demonstration of protection of CD4 T cells and DCs derived from LV-R5A32shHIV-modified HSCs against X4 and R5 infections. The effect of the anti-HIV LV is examined using CD4 T cells and DCs derived from the LV- transduced HSCs. The HSC-derived mature CD4 T cells and immature DCs will be challenged with X4 and R5 virus and virus replication will be monitored by p24 ELISA and flow cytometry using antibody against HIV antigens. Cell-to-cell transmission of virus by the LV-transduced HSCs is also examined. LV-modified HSCs are developed into mature CD4 T cells and DCs and infected with X4 and R5 virus. The infected cells are cocultured with CD4+ Molt3 cells, which are highly susceptible to HIV-1 infection. The coculture is monitored for two weeks to see if the Molt3 cells are infected by HIV-1. The T cells and DCs from LV-R5A32shHIV- modified HSCs will be resistant to HIV-1 infection. However, T cells or DCs from non-transduced or control LV-transduced HSCs will be susceptible to HIV-1 challenge.

It should be borne in mind that all patents, patent applications, patent publications, technical publications, scientific publications, and other references referenced herein are hereby incorporated by reference in this application in order to more fully describe the state of the art to which the present invention pertains.

Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.

It is important to an understanding of the present invention to note that all technical and scientific terms used herein, unless defined herein, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. The techniques employed herein are also those that are known to one of ordinary skill in the art, unless stated otherwise. For purposes of more clearly facilitating an understanding the invention as disclosed and claimed herein, the following definitions are provided.

While a number of embodiments of the present invention have been shown and described herein in the present context, such embodiments are provided by way of example only, and not of limitation. Numerous variations, changes and substitutions will occur to those of skill in the art without materially departing from the invention herein. For example, the present invention need not be limited to best mode disclosed herein, since other applications can equally benefit from the teachings of the present invention. Also, in the claims, means-plus-function and step-plus-function clauses are intended to cover the structures and acts, respectively, described herein as performing the recited function and not only structural equivalents or act equivalents, but also equivalent structures or equivalent acts, respectively. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims, in accordance with relevant law as to their interpretation.

While one or more embodiments of the present invention have been shown and described herein, such embodiments are provided by way of example only. Variations, changes and substitutions may be made without departing from the invention herein. Accordingly, it is intended that the invention be limited only by the spirit and scope of the appended claims. The teachings of all references cited herein are incorporated in their entirety to the extent not inconsistent with the teachings herein.



Claims

What is claimed is: 1. A composition comprising one or more genetic components, said one or more genetic components comprising (a) at least one interfering RNA targeting endogenous CCR5; (b) at least one interfering RNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene.

2. The composition of claim 1, wherein said elements (a), (b) and/or (c) are all packaged into a vector.

3. The composition of claim 2, wherein said vector is a viral vector.

4. The composition of claim 1, wherein component (a) is miRNA targeting endogenous CCR5.

5. The composition of claim 3, wherein said viral vector is a lenti iral vector.

5. The composition of claim 1, wherein said composition comprises a first, a second and a third miRNA, wherein said first, second and third miRNA target pol, int, and vpu, respectively.

6. The composition of claim 1, wherein said genetic components are (a) and (b).

7. The composition of claim 1, wherein said CCR5delta32 gene is codon optimized.

8. The composition of claim 1, wherein said CCR5delta32 gene is not codon optimized.

9. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

10. A viral vector comprising a CCR5delta32gene; an HI promoter driven

CCR5shRNA; and/or a triple miRNA cassette against HIV-1 pol, int and vpu.

11. The viral vector of claim 10, wherein the CCR5delta32 gene is codon optimized.

12. The viral vector of claim 10, wherein the CCR5delta32 gene is not codon optimized.

13. The viral vector of claim 10 comprising an HI promoter driven CCR5shRNA; and a triple miRNA cassette against HIV-1 pol, int and vpu, without the

CCR5delta32gene.

14. A method of treating a subject who has contracted HIV or who has otherwise been exposed to HIV, said method comprising administering a therapeutically effective amount of a composition of claim 1 to said subject.

15. A composition comprising a population of cells that have been transformed to express (a) at least one intefering RNA targeting endogenous CCR5; (b) at least one interfering RNA targeting at least one of pol, int, or vpu; and/or (c) a CCR5delta32 gene.

16. The composition of claim 15, wherein said population of cells are transformed to express (a) and (b).

17. The composition of claim 15, wherein said population of cells have been transfected or transduced in vitro.

18. The composition of claim 15, wherein said cells have been transduced with a viral vector comprising a non-codon optimized CCR5delta32gene; an HI promoter driven CCR5shRNA or CCR5miRNA; and a triple miRNA intronic cassette against HIV-1 pol, int and vpu.

19. A method of treating a subject who has contracted HIV, or who has otherwise been exposed to HIV, said method comprising administering a therapeutically effective amount of the composition of claim 15 to said subject.

20. The method of claiml9, wherein said cells are autologous.

21. The method of claim 19, wherein said cells are stem cells.

22. The method of claim 19, wherein said cells are hematopoietic stem cells, or mesenchymal stem cells.

23. A method of increasing resistance to HIV in a subject, the method comprising administering a therapeutically effective amount of the composition of claim 1 or 15, or the vector of claim 10.

24. A viral vector comprising an interfering RNA targeting CCR5 and a triple interfering RNA cassette targeting HIV-1 pol, int and vpu.