KR100696410B1 - Ribozymes and Retrocombination Retroviruses Having Retroviral Packaging Sequence Expression Constructs - Google Patents

Abstract

The present invention is a synthetic, non-naturally occurring, characterized in that its sequence has a nucleotide which defines a conserved catalytic region and whose sequence is capable of hybridizing with a predetermined target sequence in the packaging sequence of an RNA virus. It relates to an oligonucleotide compound that occurs. Preferably, the viral packaging sequence is a retroviral packaging sequence and in one embodiment an HIV-1 Psi packaging sequence. The RNA virus may be HIV-1, feline leukemia virus, feline immunodeficiency virus or one of the viruses listed in Table I below. Conserved catalytic regions originate from hammerhead ribozymes, hairpin ribozymes, hepatitis delta ribozymes, RNAase P ribozymes, group I introns, or group II introns. Can be.

The present invention also relates to a combination of antisense or free ribozymes targeting the RNA virus, and a combination of antisense and ribozymes with TRA decoy, poly TAR and RRE decoy. Vectors are also described. It also describes methods of treatment and use in vivo or ex vivo .

Description

Ribozymes and Retrocombination Retroviruses Having Retroviral Packaging Sequence Expression Constructs

This application is part of US Patent Application No. 68 / 118,082, filed January 5, 1994. In this application, various publications are referred to by their authors and year in parentheses. All references are listed alphabetically after the experiment. The overall disclosures of these publications have been incorporated by reference into the application to fully describe the state of the art to which this invention pertains.

Retrovirus

Retroviruses are viruses with RNA as genomic material. Retroviruses use host cells for their replication, which stably integrate cDNA copies of their genomic RNA into the host cell genome (Miller, 1992, and Brown, 1987). The retroviral genome has long-term repeat sequences (LTRs) at both ends (5 'and 3') of the virus's proviral cDNA. This LTR consists of U3 and U5 sequences, going from 5 'to 3', and these sequences are linked by short sequences called R's.

The LRT is adjacent to short sequences required for priming of positive and negative strand DNA synthesis by reverse transcriptase. The genome also has splice donor and acceptor sequences, which are involved in the production of sub-genomic RNA species. Right next to the 5'LTR is the sequence needed to wrap the viral RNA into a virion. This sequence is called the Psi packaging sequence. This is a specific signal that is essential to ensure the packaging of the viral RNA. The majority of viral RNA consists of gag , pol and env replicating genes, each of which encodes a core protein ( gag ), an enzyme integrase, a gene that encodes a protease and reverse transcriptase ( pol ), and an outer membrane protein (env). It is a replication gene that encodes.

Retroviral infection of cells is initiated by the interaction of viral glycoproteins with cell receptors (A) (see Figure 1). Following adsorption and uncoating, viral RNA enters the target cell and is converted to cDNA by the action of reverse transcriptase, an enzyme produced by virions (B). Next, the cDNA is circular (C), double stranded cDNA, and then integrated into the genomic DNA of the host cell by the action of integrase (D). Once integrated, proviral cDNA is transcribed from the promoter in 5 ′ LTR (E). This transcribed RNA acts as an mRNA to be translated to produce viral proteins (F) or to remain immature viral RNA. This viral RNA has a Psi packaging sequence, which is essential for packaging into virions (G). Once produced, virions are released from the cells by budding from the plasma membrane (H). In general, retroviruses do not cause lysis of host cells, with the exception of HIV. Proviral cDNAs are stably integrated into the host genome and then replicated with the host DNA so that progeny cells have a provirus. Potential anti-viral agents can target this replication control point.

Human Immunodeficiency Virus (HIV)

HIV belongs to retroviruses, and its replication is outlined above. The introduction of HIV into cells, including T lymphocytes, monocytes and macrophages of HIV, is caused by the interaction of the gp120 outer membrane protein of HIV with the CD4 receptor on the target cell surface. The amino acid sequence of gp12O varies greatly from patient to patient (even in the same patient), making vaccine production very difficult (Brown, 1987 and Peterlin et al., 988). This variability seems to be related to disease progression. The main features of HIV are: i) It has an incubation period (as with other members of the lentiviral family) so that the provirus can be incubated after incorporation into the genome of the host cell. ii) HIV has a cytopathic effect on T lymphocyte target cells. HIV starts replication after the cells containing the provirus are activated. The stimuli necessary for cell activation and what are the stimuli accompanying viral replication have not been clearly identified (Brown, 1987 and Peterlin et al., 1988). Speaking of the retrovirus as a whole, the gas , pol and env gene products are translated into structural and enzyme proteins. In detail, the tat and rev gene products are translated into regulatory proteins and act as transcriptional enhancers to induce high levels of gene expression. Nef regulates viral replication levels as another regulatory gene (Jones, 1989, Greene, 1990, and Epstein, 1991).

HIV replication is most active in activated proliferating cells; When cells are activated, nuclear transcription and cell enhancer factors bind to HIV LTR, resulting in increased transcription. In terms of retrovirus as a whole, the packaging site (Psi) is a cis-acting RNA sequence present in the HIV genome that is needed to encapsulate the genomic RNA in the capsid. The formation of cores that incorporate gas proteins, pol enzymes, and viral RNA is the final stage of the HIV replication cycle. This core obtains a membrane and then leaves the cell by budding through the cell membrane (Peterlin et al., 1988, Jones, KA, 1989, Greene, 1990 and Epstein, 1991).

To date, numerous drugs for inhibiting HIV replication have been studied. The replication steps shown in FIG. 1 describe the specific drugs targeted below.

(A) virus adsorption step on target cells

Attempts have been made to use soluble CD4 to occupy a significant amount of viral receptors so that the vires can no longer bind to the cell membrane. To date, however, this attempt has not been a successful treatment strategy (Stevenson et al., 1992). Sulfated polysaccharides have been shown to be capable of inhibiting HIV infection. Sulfated polysaccharides probably do this by interfering with cell-virus fusion (McClure et al., 1992). Antibodies to HIV itself, host cell receptors or the HIV envelope and CD4 conjugated exotoxin (CDeven Conjugated exotoxin) (Stevenson et al., 1992) are another possible means of preventing the virus from entering the cell.

(B) production of cDNA by reverse transcriptase

Compounds such as azidothymidine triphosphate (AZT) have been shown to inhibit reverse transcriptase in vitro. AZT is now routinely administered to AIDS patients and also when AIDS patients undergo bone marrow transplantation, in the latter case to protect normal bone marrow from residual HIV (Miller, 1992).

(C) cDNA transition from the cytoplasm to the nucleus

It may be possible to hinder cDNA translocation through nuclear pores or to hinder nuclear transport itself, but so far it has not been successful.

(D) cDNA4 is integrated into the host cell

It may also be possible to prevent proviral cDNA from integrating into the host cell genome (Stevenson et al.). To date, no candidate compound has proven effective.

(E) Provirus transcriptional step

5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole is known to interfere with transcriptional elongation (Stevenson et al., 1992). Sense TAR analogs can also affect transcription by inhibiting the ability to bind tat proteins and thereby activate HIV (Miller, 1992 and Sullenger et al., 1990).

(F) translation stage of HIV mRNA

Antisense RNA can inhibit viral replication by binding to viral RNA (Sczakiel et al., 1992). Binding to mRNA may help to inhibit translation; Binding to immature viral RNA may also act to inhibit the RNA from being productively packaged into virions.

(C) Viral Packaging and Production Stages of Mature Virions

Proteases cause specific degradation of HIV polyproteins. This activity is essential for the production of mature, infectious virions. Several compounds, such as α , α -difluorokedon, have been found to inhibit HIV proteases, and have shown some anti-viral activity in tissue culture. However, most protease inhibitors have short serum half-life, fast biliary clearance, and poor oral availability (Debouck, 1992).

Ribozyme

Ribozymes are enzymatic RNAs that cleave RNA and represent turnover. Some ribozymes can be paired with specific target RNAs by inducing complementary sequence arms to cause cleavage at specific sites along the phosphodiester backbone of the RNA (Haseloff et al., 1988; Rossi Et al., 1992: Hampel, 1990, 0; wang, 1992). Hammerhead ribozymes are small, simple, and have the ability to maintain site-specific cleavage when incorporated into several flanking sequence motifs (Haseloff et al., 1988; Rossi et al., 1992). These characteristics make ribozymes well suited for gene suppression.

Summary of the Invention

The present invention is characterized by having a nucleotide consisting of a nucleotide consisting of a sequence defining a conserved catalytic region and a nucleotide consisting of a sequence capable of hybridizing with a predetermined target sequence within the packaging sequence of an RNA virus. It relates to naturally occurring synthetic oligonucleotide compounds. Preferably, the viral packaging sequence is a retroviral packaging sequence and in one embodiment an HIV-1 Psi packaging sequence. The RNA virus may be HIV-1, feline leukemia virus, feline immunodeficiency virus or one of the viruses listed in Table 6 below. The conserved catalytic region may be from hammerhead ribozyme, hairpin ribozyme, hepatitis delta ribozyme, RNAase P ribozyme, group I intron, or group II intron.

The present invention also relates to a multiple ribozyme targeting the RNA virus, and a combination of ribozyme and antisense targeted to an RNA virus, and the combination further comprising polyTAR, RRE or TAR decoy. will be. Also described are ribozymes or vectors with or without antisense and polyTAR, RRE or TAR decoy. Also described are treatments and methods of use in in vivo and ex vivo.

First degree. Typical retrovirus replication cycle

(A) The virus binds to a cell surface receptor on the target cell and then genomic RNA enters the target cell after fusion and viral exfoliation.

(B) Reverse transcription occurs and viral RNA is converted to cDNA.

(C) The cDNA enters the nucleus and turns into a prototype.

(D) The cDNA is then integrated into the host cell genome.

(E) Proviruses are transcribed to produce viral RNA and mRNA.

(F) Translation occurs to produce viral proteins.

(G) A viral core is formed with the protein encoded in the virus and the viral RNA is packaged.

(H) The core obtains a membrane and leaves the cell by budding through the cell membrane.

2nd degree. Ribozyme target site in the Mo-MLV Psi packaging site The Mo-MLV / 5 ′ site represents a BalI / BalI fragment (nt 212 to nt 747) of pMLV-1 (as defined herein).

Arrows indicate ribozyme target sites that were all GUC residues.

3rd degree. Moroni mouse leukemia virus

The genome of MoMLV consists of the replication genes gag , pol and env and 5 'and 3' long-term repeats (LTRs). Viral RNA requires a Psi packaging site for packaging into virions.

4th. Anti-Mo-MLV and Anti-HIV Packaging Site Fabrication

The standard expression construct was based on pSV2neo, which had an SV40 promoter upstream of the neo ^r gene and either the antisense sequence (anti-Psi) or the designed ribozyme complementary to the Psi packaging sequence at the Sma I site of neo ^r . Is inserted.

5th. Target HIV Packaging Site

5 is a simplified illustration of the HIV genome where ribozyme 9 targets a sequence in the Psi packaging site.

6th. MO-MLV packaging region created in vitro

Cut with the bit by the ribozyme of RNA. Lane 1 is pBR322 marker DNA digested with Hinf I. Lane 2 is about 550 kb of substrate. Lanes 3, 5, 7 and 9 were Rz 243, Rz 274, Rz 366 and Rz-M7 made in vitro , respectively. The following ribozymes were added to the target substrate RNA: lane 4, Rz 243; Lane 6, Rz 274; Lane 8, Rz 366; Lane 10, Rz-M7. Cleavage reactions were performed using 10 mM Tris. Heated at 80 ° C. in Cl, pH7.5 for 2 min, then 10 mM MgCl ₂ , 50 mM Rris. 30 min at 30 ° C. in Cl, pH7.5.

7th. Southern hybridization of DNA from cell lines transfected with ribozymes or antisense constructs

Lane 1, pSV243; Lane 2, pSV 274; Lane 3, pSV366, lane 4, PSV-M7; Lane 5, pSVAs-Psi; And genomic DNA (10 μg ) of 3T3-MO-MLV cells transfected with various constructs of lane 6, pSV2neo vector alone, were digested with HindIII / NruI. Then, they were separated on a 0.6% agarose gel and then blotted onto nitrocellulose filters and hybridized with ³² P-labeled neo ^r gene probes. Arrows indicate the expected size of the neo ^r gene only (1.34 kb) and the expected size of the neo ^r gene + single ribozymes (1.38 kb), the expected size of the neo ^r gene + complex Rz (1.98 kb), or the neo ^r gene + antisense Shows the expected size (1.89kb).

8th. RNase Protection Assays to Study Ribozyme / Antisense Construct Expression 20 / μg of total RNA from a series of transfected cells were analyzed using ³² p-labeled riboprobes to investigate the expression of ribozyme or antisense constructs. . Lane 1, size marker terminal labeled DNA fragment of pBR322 digested with Hinf I; Lane 2, riboprobe of RzM7 hybridized with yeast DNA and digested with RNase; Lane 3, riboprobe of RzM7 hybridized with yeast RNA and not digested with RNase; Lanes 4-8, hybridized with RNA from cells transfected with pSV243, pSV274, pSV366, pSV-M7 and pSVAs-Psi, respectively, and digested with RNase of Rz243, Rz274, Rz366, R2M7 and As-Psi Riboprobes; Lanes 9-13 riboprobes only RzM7, Rz243, Rz274, Rz366 and As-Psi. One clone of each construct was used for analysis, which best inhibited Mo-MLV replication.

9th. Autoradiograph of Dot Blots of Viral RNA Derived from Different Mo-MLV-producing 3T3 Cells

Viral RNA samples diluted at the ratio of 1: 1, 1: 5 and 1:10 were examined with ³² P-labeled riboprobes complementary to the Mo-MLV Psi packaging site as described above. Lane 1, yeast RNA; RNA from supernatants of 3T3-MO-MLV cells transfected with lanes 2-7, pSV243, pSV274, pSV366, pSV-M7, pSVAsFsi and PSV2neo. In vitro , the RNA levels are lowered by ribozymes and antisenses that effectively cleave RNA target molecules.

10th. P24 levels during long term assay

The histogram chart shows HIV replication data measured by p24 levels at 8, 13 and 22 for ribozyme 9 (Rz-9). Rz-9 is a ribozyme construct intended to be targeted to HIV packaging sites. Vector is a control construct. Rz-9 and Rz-M are two ribozyme constructs targeted to target the tat gene of HIV. R3-M is a multi-ribozyme containing several ribozymes intended to target other sites in tat . This includes the target site of Rz-2.

Crab 11 degrees. Anti-HIV-1 tat ribozymes are designed according to sequence data of HIV-1 SF2 isolates from Genebank (LOCUS.HIVSF2 (G)). Target sites are GUC (Rzl), GUA (Gz2), GUC (Rz3) and CUC (Rz4).

12th. tat conservation sequence

Figure 13. Comparison of various tat target sequences

14th. Clonal cell line (pSV2neo, meo-a) containing a transfected clonal T cell line (SupT1) (a, d, f) vector showing protection at Rz2 and a clonal cell line (Ran a) containing any control , b, c, d, e, f)

15A-15C. Schematic (not shown to scale) of retroviral vector constructs and their resulting expression. 15A, ribozyme construct RRz2; 15B, antisense product RASH5; 15C, Polymer-TAR Product R2OTAR. Parental retroviral vectors are shown in parentheses. See the text for details.

16A-16C. Expression of Retroviral Constructs in Transduced PBLs.

Expression of ribozyme (FIG. 16A), antisense (FIG. 16B) and 20TAR RNA (FIG. 16C) was examined by Northern analysis using the corresponding ³² P-labeled probe. Please refer to the text for details.

17A-17C. Inhibition of HIV-1IIIB replication in transduced pBLs.

The transduced and selected PBLs were attacked and analyzed as described in the Materials and Methods section. Data shown are the average of five donors (FIG. 17A, ribozyme construct and FIG. 17B, antisense construct) and the average of two donors (FIG. 17C, polymeric-TAR construct).

18A-18C. Resistance of transduced PBL to infection by clinical isolate 82H.

PBLs were transduced into ribozyme constructs (FIG. 18A), antisense constructs (FIG. 18B), and polymeric-TAR constructs (FIG. 18C) and infected with 82H, as described in the Materials and Methods section. Data shown is the average of two donors.

19. Proliferation data of transduced PBLs are shown as mean ± SD (n = 3). PBL alone is PBL without transduction; LXSIN, P-20TAR, LNHL, RASH-5, LNL6 and RRZ2 are PBLs transduced with the corresponding retroviruses.

20th. CEM T4 T-lymphocyte cell lines were transduced with virus and subjected to G418 selection. Populations collected together have cells with arbitrary integrants and varying product expression levels. These cells again attacked with HIV-1.

21A-21B. RzM is a composite ribozyme directed against tat and RRzpsi / M is a ribozyme directed against packaging / compound ribozyme for tat .

The present invention provides a non-naturally occurring synthetic oligonucleotide having a nucleotide consisting of a sequence defining a conserved catalytic region and a nucleotide consisting of a sequence capable of hybridizing with a predetermined target sequence in a packaging sequence of an RNA virus. Relates to a peptide compound. Preferably, the viral packaging sequence is a retroviral packaging sequence and in one embodiment an HIV-1 Psi packaging sequence. The RNA virus may be HIV-1, feline leukemia virus, feline immunodeficiency virus or one of the viruses listed in Table 6-7 below. The conserved catalytic regjon is a hammerhead ribozyme (Haseloff et al., US Pat. No. 5,245,678: Rossi et al., US Pat. No. 5,249 796), a hairpin ribozyme (Hampel et al., European patent application) No. 89117424, filed Sep. 20, 1989), Hepatitis delta ribozyme (Goldberg et al., PCT International Application Nos. WO9l / O4319 and WO91 / 04324, published April 4, 991), RNAase P ribozyme (Altman, US Patent No. 5,168,053), group 1 intron (Cech et al., US Pat. No. 4,987,071), or group II intron (Pyle, 1993).

In one embodiment, the compound has the following structure (SEQ ID NO: 1):

You can have

Provided that each X in the structure is the same or different and represents a nucleotide whose sugar, phosphate or base may be modified or substituted;

Each A, C, U and G represents a ribonucleotide whose sugar, phosphate or base may be unmodified, modified or substituted;

3'-AAG... … AGUCX-5 'defines a conserved catalyst zone;

Each (X) _n A and (X) _{n '} defines a nucleotide consisting of a sequence capable of hybridizing with a predetermined target sequence in the packaging sequence of the RNA virus;

Each ★ represents base pair formation between nucleotides located on either side of it;

Each solid line represents a chemical bond that provides a covalent bond between nucleotides located on both sides thereof;

Each dashed line indicates, independently of each other, a unity bond that provides a covalent bond between nucleotides located on either side thereof, or any absence of such chemical bonds;

a represents an integer defining the number of nucleotides, but can be 0 or 1, if 0, then A at 5 'of (X) a is bound to X at 3' of (X) a ,

Each m and m 'represent an integer equal to or greater than 1;

(X) b denotes an oligonucleotide and b denotes an integer equal to or greater than 2.

Alternatively, the compound has the following structure (SEQ ID NO: 2):

(Wherein 3′-AAG …… AGUCX-5 ′ in the structure defines a conserved catalytic region; and none of the nucleotides (X) m forms a Watson-Crick base pair with any nucleotide in the compound) ).

In another embodiment, the compound has the following structure (SEQ ID NO: 3):

( _Wherein 3 '(X) _p4 ... (X) _p1-5 ' in the structure define a conserved catalytic region; each of (X) _F4 and (X) _F3 is a sequence whose sequence is Defining nucleotides capable of hybridizing with the target sequence;

Each solid line represents a chemical bond that provides a covalent bond between nucleotides located on both sides thereof; F3 represents an integer defining the number of nucleotides in said oligonucleotide, provided that F3 is equal to or greater than 3;

F4 represents an integer defining the number of nucleotides in the oligonucleotide, provided that F4 is from 3 to 5; Each (X) _p1 and (X) _p4 represents an oligonucleotide having a predetermined sequence such that (X) _P4 forms a base pair with 3-6 bases of (X) _p1 ;

P1 represents an integer defining the number of nucleotides in the oligonucleotide, provided that Pl is 3 to 6 and the sum of Pl and F4 is 9;

Each (X) _P2 and (X) _P3 represents a nucleotide having a predetermined sequence such that (X) _P2 will form a base pair with at least three bases of (X) _P3 ;

Each dotted line represents a chemical bond that provides, independently of each other, a covalent bond between nucleotides located on either side thereof, or indicates the absence of any such chemical bond;

(X) _L2 represents an oligonucleotide that may or may not be present, provided that L2, if present, represents an integer equal to or greater than 3).

In another embodiment, the nucleotide whose sequence defines a conserved catalytic region is derived from a hepatitis delta virus conserved region. Alternatively, the nucleotide that the sequence defines a conserved catalytic region may contain an NCCA sequence at its 3 'end. Have

The invention also relates to multiple compounds or ribozymes having the same or different conserved catalytic regions that target certain target sequences that may be the same or different. In this embodiment, each domain has two or more domains, which may be the same or different, and each domain is capable of hybridizing with a nucleotide whose sequence defines a conserved catalytic region and whose sequence can be hybridized with a given target sequence within the packaging sequence of the RNA virus. It is characterized by having a nucleotide.

The compounds may also be covalently linked to an antisense nucleic acid compound capable of hybridizing with a predetermined sequence that may be the same or different than the oligonucleotide compound in the RNA virus.

In one preferred embodiment, the nucleotides can hybridize with 243, 274, 366 or 553 target sequences in the MoMLV and site 749 in the HIV packaging site.

The oligonucleotide compound may further have at least one additional synthetic non-naturally occurring oligonucleotide compound or antisense compound that targets another gene of the RNA viral genome and is covalently linked to the gene. If the RNA virus is HIV, other regions of the HIV human genome are long-term repeat, 5 'untranslated region, splice donor-acceptor region, primer binding site, 3' untranslated region, gag , pol , protease, Integrase, env, tat , rev, nef, vif, vpr, vpu, vpx, or tev region can be selected from the group consisting of. Preferably said first nucleotide can hybridize with a 243, 274, 366 or 553 target site or combination thereof in MoMLV and site 749 in an HIV Psi packaging site and the nucleotide of said second additional compound is HIV tat Hybridization with 5792, 5849, 5886, or 6042 target sites or combinations thereof within the region.

Additional targets can be found in the HIV genome (Table III) (SEQ ID NOS: 4-7), in particular in the tat sequence and the Psi packaging region (HIV-1 SF2)

Or in Table III. Particular ribozyme sequences used herein are Rz-2, R2-M and Rz-Ø. Anti-HIV ribozyme sequence Rz-2 (single anti- tat ).

Cleavage of HIV RNA by ribozymes is a potentially useful approach. In therapeutic terms, it has several important properties.

Iii) specificity,

Ii) the ability to target a relatively large number of potential sites,

Iii) no toxicity to cells,

Over) turnover of ribozyme molecules

Iii) the variety of applicable methods of delivery and

Viability of various production methods:

a) chemical synthesis (for short molecules),

b) biochemical production by in vitro transcription, and

c) Produced as a bit as a promoter driver from integrated production.

The present invention utilizes anti-packaging site (Psi) ribozymes to inhibit HIV replication. This activity will act in the A, E, F and G phases. Disconnecting here means that the virus enters the target cell.

Ii) production of viral RNA

Iii) translation of viral mRNAs into viral proteins, and

Vi) can have an inhibitory effect on the packaging of viral genome RVA into virions.

Psi Packaging Sequence

Psi packaging sequences are cis-acting viral genome sequences that are essential for forming the protein coating of viral RNA into virions (Aronoff et al., 1991).

Packaging RNA to make viral particles is highly specific, a phenomenon known to occur at Psi sites.

The location of the packaging sites of both Mo-MLV and HIV-1 could be confirmed by functional deletion, ie by removing certain sequences and observing whether the viral RNA locus packaging process continues.

The sequence is found in the 5 'untranslated region of the retrovirus and is found to be absent in the unpackaged RNA.

In this invention, it was concluded that in order for the RNA to be packaged to be easily recognized, the packaging sequence must be exposed, accessible and recognizable.

Studies on Mo-MLV and HIV-1 packaging signals have shown that there is a stable secondary structure that is conservative in both cases (Alford et al., 1992 and Harrison et al., 1992).

According to the inventor's review, these features make the Psi packaging site a clear target of ribozyme action.

One study using antisense for retroviral packaging sequences shows that replication of Moloney murine leukemia virus Mo-MLV can be inhibited by interference of Psi sequences in transgenic animals. (Han et al., 1991).

Moroni Mice Leukemia Virus (Mo-MLV) and Human Immunodeficiency Virus (HIV-1)

Mo-MLV is a wild type mouse retrovirus without an oncogene (Figure 3) (Teich et al., 1985).

This causes leukemia by insertional mutagenesis in mice.

We used Mo-MLV as a first step in determining evidence of factors for the efficacy of anti-viral ribozymes.

Mo-MLV is a typical retrovirus that replicates along the line outlined in FIG. 1 and packaging is via the Psi packaging site.

In one embodiment of the invention, anti-Mo-MLV ribozymes targeted to Psi packaging sites and cloned in expression vectors are used to test the ability to reduce virus production in cell culture.

HIV-1, the active principle of acquired immunodeficiency syndrome (AIDS), causes cell death in T-lymphocytes (McCune, 1991; Levy, 1993).

These cells are essential contributors to the immune response.

In any anti-HIV assay, it is essential to substantially reduce or inhibit viral replication before the immune system is incapacitated due to the loss of these cells.

There is currently no effective cure for AIDS.

However, by reducing viral titers, there is an expectation that the disease can be gradually improved and further eradicated.

The development of anti-HIV-packaging sequence ribozymes is a viable way to substantially inhibit and even stop virus proliferation.

Anti-HIV- gag ribozymes have been developed before and have been shown to reduce gag- RNA and p24 levels in cells expressing ribozymes (Sarver et al., 1990).

Hammerhead ribozymes have been developed to block the translation of integrase proteins by cleaving HIV-1 integrase RNA in E. coli (Sioud et al., 1991).

Several populations have also shown that ribozymes that cleave HIV-1 RNA in U5, the 5 'untranslated region of HIV-1, or tat can protect T-cells from HIV-1 (Dropulic et al. , 1992; Ojwang et al., 1992; Lo et al., 1992; and Weerasinghe et al., 1991).

In another preferred embodiment of the invention, the anti-HIV ribozyme is targeted to the Psi packaging site and cloned in the same expression vector as in the anti-Mo-MLV construct.

This construct was also tested for its ability to reduce virus proliferation in cell culture.

Delivery of Expression Constructs

The main means of introducing expression constructs into target cells is transfection, including electroporation, liposome-mediated and retrovirus-mediated gene transfer.

Term Definition

As used herein, “Psi packaging site” refers to a site adjacent to the 5 ′ LTR that participates in encapsidation of the viral RNA to make it a virion.

As used herein, a "complementary arm" is a sequence attached to a core hammerhead ribozyme that allows binding to a specific site of a target RNA.

As used herein, “ribozyme” is a hammerhead hairpin, hepatitis delta, RNase P, group I intron or group II intron capable of cleaving target RNA.

Hammerhead ribozymes are the subject of Haseloff and Gerlach's publications (Haseloff et al., 1988) and many subsequent experimental papers.

details

The present invention relates to the treatment of viral diseases, in particular AIDS, in which the pathogenic factor has RNA as the genomic material, which RNA is packaged into virions.

The approach is to inhibit viral replication by destroying viral RNA at the Psi packaging site, a recognition sequence essential for packaging viral genomic RNA.

Cutting of this site may include: i) entry of the virus into the target cell, and subsequent integration of the provirus into the host genome, ii) production of viral RNA, iii) virus of the viral mRNA. Translation into proteins and iv) packaging of viral genomic RNA into virions.

In one embodiment of the invention, certain expression constructs are provided, which comprise a nucleotide sequence of interest.

As one preferred expression construct, a ribozyme expression construct is provided. It can access transcription of RNA capable of binding to Mo-MLV or HIV-1 RNA by complementary lymph surrounding the ribozyme when inserted into cells such as Mo-MLV or HIV-1 infected cells. .

This complementary lymph is short and the phenomenon of cleaving RNA and thereby disrupting the activity of the RNA molecule indicates the presence of the ribozyme sequence.

The present invention has been tested in several ways.

A group of experiments shows a direct correlation between ribozyme-mediated cleavage of in vitro Mo-MLV virus Psi packaging sequences and in vivo inhibition of Mo-MLV replication.

To achieve this conclusion, three main steps were taken:

i) Demonstration of cleavage with ribozyme-mediated in vitro.

Ii) transfection of the construct comprising ribozymes into Mo-MLV infected cells.

Iii) a) integration of the product, b) flqhwkdla wpwkranf qkfgus, c) various evaluation analyzes to determine the effect of ribozyme product expression on the viral replication stage.

Similar process steps were performed for HIV.

i) Design and construction of ribozyme products.

Ii) Transfection into a human T lymphocyte line of the ribozyme containing construct.

Iii) Various evaluation analyzes to determine the effects of a) product integrity, b) ribozyme product expression, c) expression of ribozyme product on viral replication stages.

The present invention inhibits viral entry into non-infected cells by i) clipping the viral RNA as it enters the target cell. Ii) acts as a viral response inhibitor by reducing the level of the functional virus exiting the infected cell.

In both cases, in the first case the viral RNA is cleaved at entry time and twice in exit.

In the latter case, cleavage reduces RNA levels by cleaving viral and mRNA.

Cleavage serves to inhibit the packaging of viral RNA, particularly at the Psi packaging site.

Several considerations have been given to selecting targets for anti-viral ribozyme action.

The criterion used for this invention is as follows.

Viii) The target must be functionally important.

Ii) have high sequence conservation among different HIV-1 isolates in the target region.

Iii) For hammerhead ribozymes, preferably there is a ribozyme target sequence such as GUA or GUC, or related triplet GUU, CUC, etc. in the sequence (Perriman, et al., 1992).

Iii) be accessible to the target surface. For example, there should be no extensive secondary structure (Rossi et al., 1992).

Psi packaging sites met the above criteria and were selected as targets for cleavage by ribozymes.

This site is a region of RNA that i) has essential functions in the retroviral replication cycle, and ii) must be recognized and able to contact other components in order to generate packaging, providing a relatively high accessibility. Iii) conserved in different strains of the same virus.

In both strains Mo-MLV and HIV it was observed that there is a strong preservation of sequence and structure at the Psi packaging site of each virus.

On the other hand, since the function of the Psi site in each virus is the same between the packaging site of HIV and Mo-MLV, it is reasonable to assume that there is no conservative structure or sequence, but there is necessarily similarity.

The secondary structure of the viral RNA was identified and a site of Psi sulphate was selected that appears to be accessible to ribozyme action.

This was in the loop region, a single-stranded unpaired hase region of RNA.

Zuker's FOLD RNA program was used to determine the location of the non-base paired sites of the Psi packaging sequence. Ribozymes were designed to target these sites. The selected site also had a GUC sequence.

The construct used in the present invention introduced a ribozyme inserted into the 3 'untranslated region of the neomycin resistance gene (neo r). The basic configuration is shown in FIG.

Such a configuration allowed the determination of integration and expression.

The former was determined by Southern analysis, the latter by cell resistance to G418 toxicity and the RNase Protection assay. Another advantage of this design is that chimera 'hybrid RNAs with small ribozyme sequences at the 3' end of relatively large neo ^r gene messengers serve to stabilize ribozymes in cells. It was. This is extremely important because without the stability the ribozyme effect is minimized.

debate

This invention provides a basis for a method that can be used to protect animals, including humans, using ribozymes from diseases caused by retroviruses.

The basic principle of the present invention is to compete for ribozymes in the packaging site of the target retrovirus in larger genes.

Carrier genes may be optional (as in the case of the present invention) or non-selective.

The expression of relatively large carrier genes provides more stable chimeric ribozyme RNA molecules.

DNA constructs are transfected into naïve cell populations to protect the cells or introduced into viral infected cell populations to reduce viral titers.

In another embodiment, the ribozyme expression construct can also be introduced by retrovirus mediated gene transfer, thereby increasing the efficiency of introduction.

A third example of this invention is a retrovirus that possesses an anti-packaging site ribozyme.

If the retroviral vector is based on Mo-MLV, ribzyme targeted to the packaging site of HIV will not cleave the Mo-MLV packaging site due to the divergence of the sequence in the two retroviruses.

In therapeutic terms, this application includes introducing this construct into ex vivo T lymphocytes or into hematopoietic stem cells of ex vivo.

One useful approach is to bind ribozyme constructs into lymphocytes or hepatocytes through retroviral vectors such as amphotropic Mo-MLV.

Hematopoietic progenitors and hepatocytes are breast targets of gene therapy because they can be present in the bone marrow or transferred to peripheral blood. Hematopoietic progenitor cells increase both bone marrow and lymphoid cells, and true hepatocytes can increase cells of all cell lines.

Treatment may include a radiotherapy to destroy the HIV infected hematopoietic system and inject the patient with hepatocytes comprising the next ribozyme. As a result, the patient's cells become resistant to HIV.

The invention also relates to transfer vectors consisting of RNA or DNA comprising nucleotides or combinations thereof, which increase the compounds described above upon transcription.

The transfer vector can be a long terminal repeat (LTR) of HIV, an adenovirus binding transfer vector, an SV40 promoter, a Mo-MLV, or an amphoteric retrovirus.

The transfer vector may also include sequences that direct the specific organ or cell, or specific site within the cell, to involve the oligonucleotide compound.

Examples of positioning at specific sites of cells include the use of packaging signals (Sullenger et al 1993).

The invention also relates to a composition comprising the compound or transfer vector described above in a suitable carrier.

The carrier may be a pharmaceutically, veterinary or agriculturally acceptable carrier or excipient. The composition also includes a TAR decoy, poly TAR or RRE decoy.

In order to prepare a DNA sequence of the present invention in a Winnuclear or eukaryotic cell, the promoter sequence of the present invention may be eukaryotic, prokaryotic or viral.

Suitable promoters are inducible, inhibitory, and more preferably constitutive. Examples of suitable prokaryotic promoters include those capable of recognizing T4 polymerase (Malik, S. et al., J. Molec. Biol. 195: 471-480 (1987); Hu, M. et al., Gene 42 : 21-30 (1986)); T3, Sp6, and T7 (Chamberlin, M. et al., Nature 228: 227-231 (1970); Bailey, JN et al., P roc. Natl. Acad. Sci. (USA) 80: 2814-2818 (1983); Davanloo, P. et al. Proc. Natl. Acad. Sci. (USA) 81: 2035-2039 (984)); P _F and P _L promoters of the bacteriophage lambda ( The Bacteriophage Lambda, Hershey, AD, Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973); Lamda II , Hendrix, RW, Ed., Cold Spring Harber Press, Cold SPring Harbor, NY (1980)); trp, recA, thermal shock and lacZ promoter, int promoter of bacteriophage lambda, bla promoter of β -lactamase gene of pBR332, CAT promoter of chloramphenicol acetyl transferase gene of pPR325, and the like.

Prokaryotic promoters are described in Glick, BR. ( J. Ind. Microbiol. 1: 277-282 (1987)); Centiempo. Y. ( Biochimie 68: 505-516 (1986)), Watson, JD et al. ( J. Mol. Appl Gen. 1: 273-288 (1982)); TK promoter of herpes virus (McKnight. S., Cell 31: 355-365 (1982)); SV40 early promoter (Benoist, C. et al., Nature (London) 290 : 304-310 (1981) and yeast gal4 gene promoter (Johnston, SA, et al., Proc Natl. Acad. Sci. (USA) 79: 6971-6975 (1982); Silver, PA et al., Proc. Natl. Acad. Sci. (USA) 81: 5951-5955 (1984)).

In susceptible eukaryotic cells or any animal, eukaryotic promoters should be used as described above to produce vectors used to inhibit retroviral infection.

Additional regulators, such as suitable promoters and polyadenylation signals, allow for the highest expression in retrovirus-infected taxotypes to be inhibited.

Thus, for example, all of the Molecular Mouse Leukemia Virus (Mo-MLV) as well as HIV-1, HIV-2 and HTLV-2 infect lymph cells and the ribozyme constructs alone or in one of these viruses (or Or higher) transcriptional control units (promoter and adenylation signals) that provide efficient expression in hematopoietic, especially lymphoid (or tissue), for efficient expression in combination with antisense RNA complementary to the packaging sequence. ) Is selected.

As exemplified below, preferred promoters include, but are not limited to, cytomegalovirus immediate early pormotor (32), lymphotropic, used with growth hormone polyadenylation signal (33) as needed. There is a Moroni-MuLV LTR promoter for use with retroviruses.

A preferred feature of the Moroni-MuLV LTR promoter is that it has the same tissue tropism as the retrovirus.

CMV promoter is expressed in lymphocytes.

Still other promoters include the VA1 and tRNA promoters.

Metal lothionein promoters have an advantage in inducibility.

The SV40 early promoter is highly expressed in vitro in bone marrow cells.

The present invention also provides a method of transcribing the nucleotide sequence of step (a) to ligation of the nucleotide sequence corresponding to the compound into a transfer vector consisting of DNA, RNA, or combinations thereof, and (b) (a) to RNA polymerase. Next, (c) relates to a method for producing the compound, consisting of the step of recovering the compound.

The invention also relates to a prokaryotic or eukaryotic host cell comprising a nucleotide sequence that produces the compound described above upon transcription.

These cells are either animal cells or progenitor cells of all hematopoietic cell lines, hematopoietic stem cells producing semi-mature and mature cells, or commited progenitor cells producing mature cells of hematopoietic cell lines, T lymphocyte progenitor cells, immature T lymphocytes. , Mature T lymphocytes, bone marrow progenitor cells or monocytes / macrophage cells.

The invention also relates to the use of said compounds for protecting hematopoietic stem cells, progenitor cells, commited progenitor cells, T lymphocyte progenitor cells, immature T lymphocytes, mature T lymphocytes, bone marrow progenitor cells or monocytes / macrophage cells.

Furthermore, the present invention relates to a method of inhibiting / treating or protecting HIV from a patient, which is composed of introducing the transfer vector into hematopoietic cells, thereby making the cells resistant to HIV to thereby inhibit / treat or protect against HIV. will be.

The introduction is ex vivo and the cells are autologous or heterologous cells with or without myeloatlation.

In one embodiment of the invention, three single and one complex hammerhead ribozymes are designed to target different sites of the Mo-MLV Psi packaging site, and one ribozyme is targeted to one site within the HIV Psi packaging site. (See Figure 2).

Mo-MLV was used as an example of a retrovirus to determine the principal.

This factor could be applied to other retroviruses, including HIV. Experiments were also performed on HIV-1.

In the present invention, non-human animals and their progeny include cells which at least to some extent non-naturally express or retain oligonucleotide compounds.

Embryonic cells or somatic cells of transgenic non-human animal cells comprise non-naturally occurring oligonucleotide compounds in expressed form, introduced into the animal or its progenitor cells at the embryonic stage, as described in the following: Patent 4,736,866, 5,175,383, 5,175,384 or 5,175,385. See also (Van Brnt, 1988; Hammer, 1985; Gordon et al. 1987; Pittius et al., 1988; Simons et al. 1987; Simons et al. 1983).

The invention also includes a method of making a cell resistant to viral infection, comprising treating the cell with the non-natural oligonucleotides described above.

Preferably, this treatment is ex vivo.

Moreover, as used herein, the terms antisense and ribozyme also include compounds having modified nucleotides, deoxynucleotides, peptide nucleic acids, and the like.

They can be used for ex vivo treatment, ie topical treatment.

An effective amount of a non-naturally occurring oligonucleotide compound of the present invention is generally in a concentration of about 1 nM to about 1 mM in a complex form such as topical creams, sterile injectable compositions or other parenteral administration compositions.

For topical formulations, it is generally suitable to contain about 50 uM to 500 uM of non-naturally occurring oligonucleotide compounds.

Compounds having nucleotide derivatives, which are derivatives containing chemically modified groups such as phosphorothioate or methylphosphonate derivatives, are active at nanomolar (nM) concentrations.

This range of concentration is also suitable to avoid toxicity.

Therapeutic strategies, including the treatment of diseases using the compositions of the present invention, are generally the same as the antisense approach, as described in Antisense Bibliography (Chrisley, 1991).

In particular, the concentration of the compound used, the method and form of administration, and the formulation are the same as those selected for antisense applications.

As used herein, an "effective amount" is an amount that provides a desired effect in a mammal having certain conditions and dosage regimens.

The compositions of the present invention, which consist of an effective amount of a compound and a suitable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier, are useful for treatment.

The composition may be in liquid or lyophilized or other desiccant form, and may contain various buffering diluents (e.g. Tris-HC1, acetate phosphate), pH and ion enhancing additives such as albumin or gelatin to prevent surface absorption, surfactants (e.g. , Tween 20, Tween 80, Pluronic F68, bile salts, solubilizers (e.g. Thimerosal, benzyl alcohol), bulking substances or tonicity modifiers (e.g. lactose, mannitol), non-naturally occurring oligonucleotides Covalent attachment of polymers, such as polyethylene glycol to compounds, complexation with metal ions, or the incorporation of polymer compounds, such as polylactic acid, polyglycolic acid, polyvinylpyrrolidone, into particulate agent Or binding of substances into liposomes, micelles, microemulsions, mono-lamellar or multi-lamellar vehicles, erthrocyte ghosts. Include.

This composition will affect the physical state, solubility, stability, in vivo release rate and in vivo clearance of the oligonucleotide.

Antioxidants (eg, ascorbic acid); Low molecular weight (less than 10 residues) polypeptides (ie, polyarginine or tripeptides); Proteins (such as serum albumin, gelatin or immunoglobulins); Amino acids such as glycine, glutamic acid, aspartic acid or arginine; Chelating agents such as EDTA; And ingredients such as sugar alcohols such as mannitol or sorbitol.

Controlled release compositions include lipophilic depot (eg fatty acids, boxes, oils) formulations.

In addition, particulate compositions coated with polymers (eg, poloxamers or poloxamines) and non-naturally occurring oligonucleotide compounds bound to tissue-specific receptors, ligands or antigens, or to ligands of tissue-specific receptors are also included. It is included in the invention.

Furthermore, specific nucleotide sequences can be added to target non-naturally occurring oligonucleotide compounds of the invention to the nucleus, plastids, cytoplasm, or to specific forms of cells.

In another embodiment of the compositions of this invention, protective coatings, protease inhibitors or penetration enhancers are added to the particulate formulation according to various routes of administration, including parenteral, pulmonary, nasal or oral.

Suitable topical formulations include gels, creams, solutions, emulsifiers, hydrocarbon polymers, biodegradable matrices thereof, vapors, vapors, mists, aerosols, or other inhalants.

Non-naturally occurring oligonucleotide compounds can be encapsulated using a solid carrier including water, wax, film or chew gum.

Penetration enhancers to aid delivery through epithelial cells are also known and include, but are not limited to, dimethylsulfoxide and glycols.

Ribonucleotides and deoxyribonucleotide derivatives or variants are well known in the art and are available as commercially available DNA synthesizers (see Saenger, l984, in particular, pp. 159-200).

Nucleotides consist of base, sugar and monophosphate groups. Thus, nucleotide derivatives, substituents, or modifications are made at the base, sugar or monophosphate level.

Many modified bases are found in nature and a wide range of modified bases have been produced synthetically (Saenger, 1984; and CRC Handbook of Biochemistry).

Suitable bases include inosine, 5'-methylcytosine, 5'-bromo-uracil, xanthine, hypoxanthine and similar bases thereof.

Alkylation of nitrogen atoms or carbon atoms in rings such as, for example, N ¹ and N ⁷ of guanine and C ^{5 of} cytosine; Keto substitution by a thioketo group; Saturation of a C═C double bond; And amino groups and ring nitrogen atoms can be alkylated, such as the introduction of C-glycosyl bonds in pseudouridine.

6-mercaptopurine and 6-mercaptoguanine are examples of thioketo derivatives.

Bases may be substituted with various groups such as hydrogen, hydroxy, amine, alkyl, azido, nitro, phenyl.

The base may be substituted with other kinds of chemicals, such as side chains or linkers of amino acids that do not bind or bind other chemical units such as, for example, acidic or basic groups.

For example, guanine (G ₃ ) can be substituted with tyrosine and similarly cytosine (C1) or adenine (A11) can be substituted with histidine.

The sugar moiety of nucleotides is also modified according to methods known in the art (Saenger, 1984).

The present invention performs various modifications to sugar moieties of nucleotides so long as the modification does not eliminate the cleavage action of the compound.

Examples of modified sugars include substituted secondary hydroxy groups with hydrogen, amino or azido groups; 2'-methylation, glycosyl C _r is cis- oriented -N link arabinose New provide nucleoside O ₂ 'to-hydroxyl same type modified product (conformation variant), for generating a carbon α- New Clo seed Structural isomers in C _r , and the like.

Moreover, non-ribose sugar compounds such as hexose such as glucose and pentose such as arabinose may be used.

Phosphate moieties are also well known in the art and are susceptible to derivatization, ie modification.

For example, the substitution of nitrogen and sulfur in the oxygen of the carbon derivative is phosphoramidate, respectively. Phosphorothioates, phosphorodithiolates and phosphonates are produced.

The substitution of oxygen by nitrogen, sulfur of the carbon derivatives may occur at bridging or non-crosslinking sites.

The fact that phosphodiester and phosphorothioate derivatives efficiently enter cells (especially for short lengths) with regard to cell receptors is well established from studies with antisense oligonucleotides.

Methylphosphonates are easily captured by cells due to their electrical neutrality.

The phosphate moiety can be fully substituted with peptide nucleic acids (see Hanvey et al., 1992; Nielson, 1991; and Egholm, 1992).

For example, siloxane bridges, carbonate bridges, acetamidate bridges, carbamate bridges are well known to those skilled in the art (Uhlmann and Peymenn, 1990).

The following examples are intended to illustrate the invention as claimed. The invention is described in detail in the experimental section below. This is to aid the understanding of the present invention but is not intended to limit the scope of the present invention.

Example 1

In vitro ribozyme-catalyzed cleavage of Mo-MLV Psi packaging sequence.

In vitro cleavage reactions were performed prior to ribozyme testing in cell culture to demonstrate that the target site was actually cleaved.

Four sites were selected at the Mo-MLV packaging site depending on the presence of GUC bases and the potential accessibility to sites in the hypothetical RNA secondary structure inferred by the Zuker's FOLDRNA program (Zuker et al., 1981). .

These sites were designated 243, 274, 366 and 553 based on nucleotide distance from the 5'-end of the viral transcript (Figure 2).

These nucleotide positions are as described in RNA Tumor Virus (Coffin, 1985).

Two types of ribozymes were designed: three single ribozymes targeting sites 243, 274 and 366, each with 12 nucleotide lymph and four sites with intervening arms of sequence length between each target site. Complex ribozyme targeted to all.

The areas and the overall design are shown in FIG.

Single ribozymes were constructed by cloning an artificial double stranded insert with overhanging Psti and EcoRI ends into pGEM3Zf (+).

As a result, pGEM243, pGEM274 and pGEM366 plasmids were obtained.

Complex ribozymes were constructed by modifying the standard in vitro mutant protocol (Warrilow et al., 1992).

This plasmid was named PGEM-M7.

DNA sequencing confirmed successful cloning and sequence integrity.

In Bal 1-Bal I fragments of Mo-MLV derived from pMLV-1, the Psi packaging sequence was cloned into the pGEM3ZF (+) vector and transcribed to serve as a substrate for in vitro ribozyme cleavage. .

Run-off transcription mixture (50 μl) to generate ribozymes or substrates was linearized proteinase K treated template (template), 30 mM dithiothreitol, 400 μM of each rNTPs, 40 mM Tris-Cl, pH 8.0, 2 mM spermidine, 6 mM MgCl ₂ , 50 mM NaCl, [ ^a-32 P] -UTP (400-800 Ci / mmole, 10 mCi / ml) 1 μl, RNasin 1 unit and 10 units of T7 or SP6 RNA polymerase (Stratagene) were contained. After 1 hour of incubation at 37 ° C, 10 units of RNase-free DNase (Progema) was added, and the mixture was incubated at 37 ° C for 15 minutes. After extraction with phenol-chloroform, 0.1 volume of 3M sodium acetate and 2.5 volumes of ethanol were added to precipitate RNA transcripts.

For cleavage reactions, ribozyme and substrate (1: 1 molar ratio) were pre-incubated at 80 ° C. for 2 minutes and further incubated at 37 ° C. for 30 minutes in the presence of 50 mM Tris-Cl, pH 7.5 and 10 mM MgCl ₂ .

An equal volume of stop mix (8 M urea, 50 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol) was added to stop the reaction and denaturing with 8 M urea. ) 6% polyacrylamide gels were analyzed and then autoradiography was performed.

Engineered ribozymes targeting different sites of the Mo-MLV proviral packaging sequence have been shown to cleave target RNA in vitro at selected sites.

Incubation of ³² P-labeled ribozyme RNA and ³² P-labeled Psi transcript in approximately equimolar amounts for most of the ribozyme constructs was effective at mild physical conditions (37 ° C., 10 mM MgCl ₂ and 50 mM Tris-Cl, pH 7.5). The substrate could be cleaved.

A representative example of such digestion is shown in FIG.

The size of the cleaved Psi fragments produced by Rz274 and Rz366 matched the position of the cleavage expected site at 62nt + 473nt and 154nt + 381nt bands, respectively.

Complex ribozyme (Rz-M7) produced four fragments (50nt, 92nt, l87nt and 240nt) as expected with several partially truncated fragments.

In the case of Rz243, no cleavage was observed at 37 ° C., while there was a weak cleavage at 50 ° C., resulting in fragments of moderate size (data not shown).

Except for ribozyme 243, these results showed efficient site-specific ribozyme mediated cleavage.

Example 2

Anti-Mo-MLV Packaging Site (Psi) Fabrication

Following the demonstration of efficient in vitro cleavage, the 3 'ratio of the neo ^r gene bound to the simian virus 40 (SV40) early promoter is engineered ribozyme such as a long antisense sequence complementary to the Psi packaging site. It was cloned into the translation site (Figure 4).

neo ^r is a prokaryotic gene that phosphorylates neomycin or neomycin analog 418 and thereby encodes an enzyme that inactivates it.

The latter is toxic to mammals and the expression of the exogenous neo ^r gene enables cell survival.

The construct with the SV40 promoter bound to this neo ^r gene is in a mammalian expression vector, pSV2neo and is shown schematically in FIG.

Ribozyme inserts and antisense controls were cloned into the Smal region of the 3 'untranslated region of neo ^r by blunt-ended ligation.

The production vectors were named pSV243, pSV274, pSV366, pSVM7 and pSVas Psi (antisense preparation), respectively.

Example 3

Transfection of the Construct into a 3T3-Mo-MLV Producing Cell Line

Calcium phosphate transfection process table was used to transfect various pSV2neo derived constructs into 3T3-Mo-MLV cells (Chen et al, 1987).

Positive colonies formed after 9-12 days in the presence of 500 μg / ml of G418.

For each construct, 4-7 colonies were isolated using a cloning cylinder.

These colonies were propagated and stored in liquid nitrogen and later used for further analysis.

Selection of 500 μl / ml G418 for 10-14 days yielded several stable clone cell lines for each construct.

To confirm the integration of the transfected DNA expression constructs, genomic DNA was prepared from the transfected cell line and Southern analysis was performed.

Restriction enzymes HindIII and NruI were used to digest genomic DNA to produce fragments containing neo ^r + inserts (ribozyme or antisense).

Neo ^r specificity probes were then used to determine the presence of the construct.

From the Southern analysis shown in FIG. 7, it is clear that cells infected with both ribozyme and antisense constructs and selected in G418 include the neo ^r gene plus the appropriate ribozyme or antisense sequence. The size of the HindIII-NruI fragment hybridized with the neo ^r probe in each case was 1.3 kb for the neo ^r gene alone; neo ^r + 1.38 kb for single ribozyme; 1.98 kb for neo ^r + complex ribozyme; It was confirmed that the size was expected as 1.89 kb for the neo ^r + antisense sequence.

Expression of ribozymes or antisense constructs was predicted due to G418 resistance in positive transfectants.

This was further tested in transfected cells using the RNase protection assay.

Total RNA was extracted from a given cell line using guanidium thiocyanate process and 20 ug total RNA was extracted with 80% deionized formamide, 100 mM sodium citrate pH 6.4, 300 mM sodium acetate pH 6.4, 1 mM Hybridization with the corresponding ³² P-labeled riboprobe in a solution containing EDTA followed by RNase digestion of the hybridized RNAs (5 ug / ml RNase A and 10 units / ml RNaseT1).

If ribozymes were not expressed, complementary riboprobes would not be able to bind.

The RNA will then remain single stranded and all will be digested by the RNase.

The reaction mixture was separated by electrophoresis.

As shown in Figure 8, the assay results indicated that all ribozymes and antisense constructs were expressed as expected.

Protected fragments are 65 bp (single ribozyme), 588 bp complex ribozyme and 524 bp (antisense).

Example 4

The ability of the production to inhibit Mo-MLV replication.

Stable 3T3, Mo-MLV clonal cell lines transfected with different constructs were established, and Mo-MLV replication levels were evaluated by XC plaque analysis.

The XC assay is a new lithium plaque assay for Mo-MLV, based on that Mo-MLV-producing cells cause fusion of XC cells.

The titer of Mo-MLV was measured as described in Gautsch et al., 1976, except that 8 ug / ml polybrene (Sigma) was used for infection to enhance viral defects on target cells.

Supernatants of each Mo-MLV-producing cell line were added to uninfected mouse NIH3T3 cells pre-treated with 8 μg / ml polybrene one hour before infection.

After incubation for 20 hours in growth medium, infected NIH3T3 cells were incubated with XC cells in 2x2 mm ² grided plate medium.

The plate was then fixed with methanol, stained with 1% methyleneblue + 0.1% Centian violet and searched for syncytium plaques under a microscope.

To ensure that this assay was performed within the straight portion of the dose-response curve, 3.5 × 10 ⁵ cells per plate were infected with the diploid dilution virus solution and transferred to the mixed medium with XC washes after 24 hours. The results of three independent experiments are shown in Table 1.

Inhibition of 74% to 77% of composite plaque formation was observed from cells containing Rz274, Rz, 366, Rz-M7, and AS-Psi compared to pSV2neo vector-containing cells, whereas in the case of R232-containing cells there was marked inhibition. Did not look.

The experimental results are consistent with the in vitro cleavage results (Fig. 6), where even R232 did not efficiently cleave the substrate under experimental conditions.

This inhibitory effect was confirmed using viral RNA dot-blotting. A 16 hour culture of NIH3T3 virus-producing cells was centrifuged (12,000 rpm, 10 minutes, 4 ° C.) in a microcentrifuge to separate 1 ml of supernatant.

Viral RNA was precipitated in 8% PEG 8000 and 0.5 M NaCl.

After extraction with phenol-chloroform, it was blotted onto a positively charged nylon membrane (Zeta-Probe, Bio-Rad) in an alkaline transfer solution (Reed et al., 1985).

50% formamide, 5 X Denhardt solution, 0.5% SDS, 100 mg / m1 denatured herring sperm DNA and ³² P-labeled riboprobe transcribed from the T7 promoter of pGEM-Psi overnight at 42 ° C The ration was performed.

Viral DNA was measured by dot scintillation counting.

Viral RNA of supernatant from ribozyme or antisense-infected 3T3-Mo-MLV cells was measured and compared to values from pSV2neo-infected 3T3-Mo-MLV cells. As seen from FIG. 9 and Table 2 (excluding Rz243-expressing cells), the amount of viral RNA generated from all cell lines expressing ribozyme or antisense was reduced to substantially the same amount as in synthium analysis.

Following transfection of these ribozyme constructs to Mo-MLV infected cells, only ribozymes that showed efficient in vitro cleavage showed the ability to inhibit Mo-MLV replication in vivo (about 70-80%).

These results show a direct correlation between in vitro cleavage and in vivo ribozyme mediated virus inhibition.

The above experiments were the basis for a study of ribozymes designed to target HIV Psi packaging sequence sites to reduce viral titers.

TABLE 1 Cynthium plaques induced by Mo-MLV released from transfected cells.

Supernatants from RZ or AS construct-containing cells were diluted 10 ^-2 and used for infection of NIH3T3 cells.

+ Values are the average of the number of plaques from two clonal cell lines of each construct in three independent experiments. Numbers are expressed as mean ± standard deviation. Replica plates containing infected cells at the same dilution had similar synthium plaque numbers.

Table 2 Degree of hybridization for viral RNA dot blots

* cpm count was obtained from two blots at 1: 1 dilution. Viral RNA dot blot analysis was performed as described in Materials and Methods. Following radiographing, the filter corresponding to each dot was cut for liquid scintillation counting.

Example 5

Anti-HIV Packaging Site Fabrication

For ribozyme targeting, one (GUA site) was selected at the HIV-1 (HIVSF2, Levy, 1984) Psi packaging site (nuc. 735 to nuc. 765 from the 5 'end of the HIV genome).

For the previous constructs, the synthetic ribozymes were cloned into the Smal region of the 3 'untranslated region of the neo ^r gene of the pSV2neo vector by blunt-ended ligation.

DNA sequencing confirmed the success of cloning and sequence integrity.

This construct was named pSV-Rz-HIV-Psi.

This production is shown schematically in FIG.

Example 6

Transfection of pSV-Rz-HIV-Psi into T Lymphocytes

An anti-HIV packaging site construct, pSV-Rz-HIV-Psi, was electroporated into Sup T-1 cells, a human T lymphoma cell line.

Exponentially proliferating cells were harvested and viable cell number was measured by dye exclusion.

Cells were washed with PBS and resuspended at a concentration of 1 × 10 ⁷ viable cells / ml in RPMI medium containing 10 mM dextrose and 0.1 M dithiothreitol while not containing FCS.

Electroporation was performed in 0.4 cm cuvette (Bio-Rad) using 0.1 ml of cell suspension and 10 μg of pSV-Rz-HIV-Psi plasmid DNA.

The cell and DNA mixtures were subjected to a single pulse of 960 uF and 200V with a Gene Pulser (Bio-Rad). After the impact, the covet was incubated for 10 minutes at room temperature, then the cells were transferred to 10 ml of RPMI medium containing 10% FCS and placed in an incubator (5% CO ₂ , 37 ° C.).

After 48 hours of electroporation, cells were selected in medium supplemented with 800 μg / ml G416.

After 9-12 days, positive colonies were isolated and propagated to cloned isolates and used for HIV protection assessment analysis.

Example 7

Evaluation of the Protective Ability of Ribozyme-expressing Constructs Against HIV Infiltration

To evaluate the efficacy of anti-HIV Psi ribozyme constructs in cell culture, two assays, p24 and synthium formation were performed.

The HIV-1 p24 antigen assay is an enzyme immunoassay that uses mouse monoclonal antibodies against HIV core antigen coated on a microwell strip.

The HIV-1 synthium assay is based on the observation that HIV-1 fuses with target T lymphocytes and interacts to form syntium, a large cell containing many nuclei.

Clonal ribozyme-product expressing cells and controls at m.o, j. From 0.1 to 1 were infected with HIV-1 (SF2).

After 2 hours, cells were washed and 10 ml of fresh medium was added.

Every 3-4 days, synthium and viable cell numbers were measured.

For syntium formation, about two log higher doses were required to achieve the same results as in the control group without ribozyme (Table 3).

Moreover, the presence of ribozymes resulted in the delay of synthium formation (Table 4).

In another experimental process table, cells are pelleted and a partial sample of the supernatant is taken for p24 analysis.

Representative results are shown in FIG.

This experiment shows p24 level inhibition 22 days after challenge.

On days 8 and 13 post-infection, 80% inhibition is observed in ribozyme-expressing cells compared to cells containing only the vector, while at day 22 about 60% inhibition is observed.

This result provides evidence for the fact that HIV replication can be inhibited by adding ribozymes for Psi packaging sites into T lymphocytes.

Table 3. New Sithium Formation

HIV-1 (SF33) was used for infectivity analysis (m.o.i. 0.1-1)

Table 4. Synthium Formation of Infected SupT1 Cells

Count the number of synthium of each culture in four low-power fields, no synthium formation,-; 1-5 synthium, +; 6-10 Cynthium, ++, 10 or more Cynthium, +++.

Mock is uninfected SupT1 cells.

Example 8

In addition to targeted φ , the efficacy of other ribozyme constructs was also evaluated.

Unexpectedly, it was found that single ribozymes targeted to sequences in the tat gene (Rz2) of HIV-1 were also effective in inhibiting HIV replication.

Sequence conservation of this tat site was assessed and found to be very conservative among other HIV-1 isolates (see Figure 12).

This is the first experiment with this tat sequence as an HIV-1 ribozyme target site.

13 shows a literature report of tat target sites targeted by other researchers to site 1 (Crisell et al., 1993) and site 3 (Lo et al., L992 and Zhow et al., 1992). It is becoming.

summary

RRz2, an LNL6-derived virus with a ribozyme that targets the HIV tat gene transcript inserted into the 3 'region of the neomycin resistance gene (neo ^r ); RASH-5, an LNHL-derived virus comprising an antisense sequence to the 5 'leader region of the HIV-1 downstream of the human cytomegalovirus (HCMV) promoter; And human peripheral blood with various recombinant retroviruses, including R20TAR, an LXSN-derived virus with 20 tandem copies of HIV-1 TAR sequence driven by Moroni mouse leukemia virus gut terminal repeat (LTR). Lymphocytes (PBLs) were transduced.

After G418 selection, transduced PBLs were challenged with HIV-1 laboratory strain IIIB and primary clinical isolates.

The results show that PBLs from different donors were transduced and they are resistant to HIV-1 infection.

For each construct, the p24-antigen level was reduced by about 70% compared to PBLs transduced with the corresponding control vector.

Molecular analysis showed histological expression of all transduced genes from retroviral LTR, while no transcript was seen from the internal HCMV promoter for antisense constructs.

Transduction of PBLs and thus transgene expression did not affect surface expression (measured by flow cytometry) or cell proliferation (tested with [ ³ H] tmidinc uptake assay) of CD4 ⁺ / CD8 ⁺ . .

These results indicate the potential utility of these anti-HIV-1 gene therapeutics and show the pre-clinical value of this PBL assay system.

Human immunodeficiency virus (HIV) has been identified as an etiological factor in AIDS and related diseases (Barre-Sinoussi, F. et al., 1983; Gallo, RC et al., 1984). .

At present, there is no suitable treatment for this disease, and the use of genetic manipulations to suppress HIV replication appears to be a new and trustworthy study of AIDS treatment.

Possible gene therapeutic approaches to interfere in terms of HIV-1 replication include ribozyme expression that catalytically inactivates HIV-1 RNA by cleavage, antisense RNA expression that inhibits reverse transcription, progression and translation of HIV RNA; Expression of mutant HIV structures or regulatory genes with dominant inhibitory activity; And those that utilize expression of RNA decoy that inhibits HIV-1 transcription, progression and packaging.

In reports published to date, the transport method of choice for the introduction of transgenes and gene therapeutic anti-HIV-1 factors is retroviral vectors.

This vector has been tested in human hematopoietic T lymphocyte lines, such as CEM, SupT1 and MOLT-4 (Sarver, N. et al., 1990; Weerashingee, M. et al., 1991; Yu, M. et al. , 1993; Yamada, O. et al., 1992;, Rhodes, A. and James, W., l991; Sczakiel, G. et al., 1992; Lisziewicz, J. et al., 1991, 1993; Trono, D. et al., L989; Malim, MH et al., 1992), in vitro assays have several desirable characteristics, including non-limiting growth capacity, while having the disadvantage of being typical cells.

It is therefore necessary to test the efficacy of anti-HIV gene therapy reagents in human primary cells such as peripheral blood lymphocytes (PBLs).

In these cells, the primary target cell for HIV infection is CD4 ⁺ sub-population and it is this cell population that is primarily deficient in AIDS patients.

However, there are currently no reports that primary PBL assays have been used for anti-HIV gene therapeutic approaches.

We have: i) tested anti-HIV factors including ribozymes, antisense and RNA TAR decoy, and ii) PBL transduction, G418 selection and HIV- using both laboratory and clinical HIV-1 isolates. A comprehensive study of human PELs was conducted to establish the conditions for the attack.

This experiment is ribozyme targeted to HIV-1 tat gene; Antisense sequences complementary to the 5 'leader region of HIV-1; Or transduction of primary PBLs with retroviral constructs expressing 20 TAR RNA decoy confers substantial resistance to HIV-1 infection.

This assay system is an improvement over previous analysis of anti-HIV retroviral constructs and serves to complement local T cell line assays.

By using this system, we have generated significant data on the clinical validity of HIV gene therapy.

Materials and methods

Cell Lines: Packaging cell lines φ 2 (Mann, R. et al. 1983) and PA317 (ATCC CRL 9078) were cultured in Dulbecco's modified Eagle's median (DME) containing 10% fetal bovine serum (FBS).

RA317 cells were selected every 6 weeks (5-7 days) in HATT medium).

Ψ CRE and Ψ CRIP (Danos, O. and Mulligan, RC 1988) were grown in DME ÷ 10% calf serum (BCS).

Retroviral Vector Construction: A chemically synthesized hammihead ribozyme targeted to the HIV-1 tat gene transcript (nt 5865 to nt 5882 of HIV-1 IIIB, GGAGCCAGTAGATCCTA), within the 3 'untranslated region of the neomycin resistance gene It was cloned into the SalI site of the LNL6 vector (Bender, MA et al. 1987) (Figure 15 A).

This production was named RRz2.

For antisense constructs, a pNHP-1 vector was obtained by removing a human HPRT cDNA from the BamHI site with a 550 bp BamHI fragment of an HXB2 clone partially comprising the R, U5 and 5 'portions of the gag gene (nt 78 to nt 628). From (Yee, JK et al, 1987) into the BamHI site of the LNHL vector in the antisense direction (FIG. 15B).

The resulting antisense construct was named RASH5.

20TAR fragments were inserted into the XhoI and BamHI sites in the LXSN vector to prepare a polymeric-TAR construct (Miller, A. D. et al. 1989) and named R20TAR (FIG. 15C).

Preparation of Amphoropic Retroviruses: Retroviruses LNL6 and RRz2 were prepared by trans-infection comprising packaging cell lines Ψ 2 and PA317 cells.

About 80% of confluent Ψ 2 cells were transfected with 10 μg / ml of the construct DNA by using calcium precipitation and incubating in DME medium containing 10% FBS (DME growth medium) for 14 hours.

This medium was then removed, replaced with fresh DME growth medium and incubated overnight at 37 ° C., 5%, CO ₂ .

Ecotropic virus supernatants were then collected from transfected Ψ 2 cells and used to infect second-fusion (60-80%) PA317 cells in DME growth medium in the presence of 4 μg / ml of polybrene.

After 24 hours incubation at 37 ° C., 5% CO ₂ , infected PA317 cells were trypsinized and added at a ratio of 1:20 into DME growth medium containing 750 μg / ml G418.

The medium was changed every 3-4 days until colonies formed.

10-29 colonies for each construct were obtained and expanded for viral titer (neomycin resistance colony analysis) and replication-competent retrovirus (RCR) analysis (Miller, A. D. and Rosma, G. J. 1989).

Retrovirus LNHL, RASH5, LXSN and R20TAR were prepared by transfecting Ψ CRE and Ψ CRIP cells.

20-96 colonies were isolated from each construct and used for titer and RCR analysis.

Transduction of Human PBLs: Peripheral blood mononuclear cells (PBMCs) were prepared from leukopack of healthy donors by Ficoll-Hypaque gradient centrifugation.

CD4 ⁺ cells were enriched by depletion of CD8 ⁺ cells using MicroCELLector Flask (Applied Immune Science) according to producer instructions.

5 μg / ml of phytohemagglutinin (PHA, in RPMI-1640 medium supplemented with CD4 ⁺ enhanced PBLs (5 × 10 ⁵ cells / ml), 10% FBS and 20 recombinant units of human recombinant intileukin 2 (PHA, Sigma) or 10 ng / ml of OKT3 monoclonal antibody (Janssen-Cilag) for 48-72 hours.

Transduced (using m.o.i. of 0.5) stimulated PBLs by exposing the cells to a producer cell-free retrovirus stock for 18 hours in the presence of 4 μg / ml polybrene.

After 48 hours of transduction, PBLs were selected for 10-14 days in RPMI growth medium containing 300-500 μg / ml G418.

PBLs were recovered for one week in fresh RPMI growth medium without G418 and then used for the attack of HIV-1.

HIV-1 Infection: Infection titer (TCID50) of HIV-1 laboratory strain IIIB and clinical isolate 82H was measured in human PBLs as described in (Johnson, V. A. et al. 1990).

⁵ × 10 ⁵ transduced PBLs were sensitized with 100 TCID50 HIV virus at 37 ° C. for 2 hours and then the cells were washed twice with RPMI-1640 and the cells were resuspended in 5 ml RPMI growth medium.

Every 3-4 days, supernatant aliquots were taken as samples for the p24 antigen ELISA (Coulter).

RNA Analysis: Total cellular RNA was extracted from the transduced PBLs using the guanidium-isothiocyanate method (Chirgwin, J. J. et al. 1979).

15 μg of RNA was fractionated on 1% agarose-formaldehyde gel, transferred to nylon membrane (Hybond-N), ³² P-lebelled neo ^r -specific probe, 550 bp BamHI fragment of HIV-1 HXB2 or 20 TAR Hybridization with fragments examined neo ^r -ribozyme, antisense and TAR expression, respectively.

FACS analysis of transduced PBLs: 1 × 10 ⁵ transduced PBLs were CD4 or CD8 specific fluorescein isothiocyanate (FITC) -conjugated monoclonal antibody (Becton Dickinson) or control antibody (FITC- mouse IgGl, Becton-Dickinson) were incubated at 4 ° C. for 20 minutes.

After washing twice with PBS, cells were analyzed on Becton Dickinson FACScan.

Proliferation Assay: PBLs were transduced as described above.

After selecting from G418 and recovering in fresh RPMI growth medium, viability was assessed by trypan blue exclusion and cell number adjusted to 1 × 10 ⁶ viable cells / ml.

1 × 10 ⁶ cells were inoculated in triplicate wells (Corning 24 well-plate) and 1 μCi of 6- [ ³ H] -thymidine (5Ci / mmol, Amersham) was added to each well.

After 48 hours of incubation, the cells were transferred to a glass fiber filter under vacuum, washed three times with ice-cold phosphate buffered saline solution and precipitated with 3 × 5 ml ice-cold 10% trichloroazate acid (w / v).

The filter was rinsed with ethanol and β -scintillation count was performed.

Statistical analysis was performed using Student's t-test.

result

Development of high-titer amphoteric retroviruses containing various transgenes.

Three different retroviruses expressing transgene (ribozyme, antisense or polymeric-TAR) were constructed based on different vector roots.

RRz2 was constructed by inserting an anti-HIV tat ribozyme into the 3 'untranslated region of the neo ^r gene induced by the MoMLV LTR (long terminal repeat) of the LNL6 vector (FIG. 15A).

Chimeric RNA transcripts containing both neo ^r and ribozyme genes are expected from this retrovirus.

For RASH5 retroviruses (FIG. 15B), antisense sequences can be transcribed from viral LTR or internal human CMV promoters.

In the R20TAR construct, polymeric-TAR is expressed from viral LTR (Figure 15 C).

These three retroviral constructs and their corresponding control vectors were used to generate the amphoteric producer cell line.

Virus titers ranged from just 10 ⁵ to 5 × 10 ⁶ cfu / ml, as measured by standard milestones (Miller, AD and Rosma, GJ et al., 1989).

In general, at least 10 ⁶ retroviral titers were used for transduction experiments.

All virus lines were tested to confirm that there was no RCR and stored at -80 ° C.

Retroviral transduction of PBLs.

To optimize PBLs stimulation for retroviral transduction, CD4 ⁺ enhanced PBLs were compared to responses to PHA or OKT3 antibodies.

No differences in cell multiplication time, viability and transduction capacity were observed in culture with PHA or OKT3.

In this experiment, OKT3 antibodies were used because they are licensed for human use.

Stimulated PBLs were then transduced with an amphoteric retrovirus using 0.5 m.o.i.

Relative transduction efficiency was determined based on cell number surviving G418 selection.

Overall transduction efficiency varied from 2-7% depending on the donor blood pack.

G418 selection of transduction PBLs was an important step in PBL analysis.

A two-step process was adopted to get the perfect selection.

For each batch of PBLs, a C418 toxicity dose assay was set up and at the same time a G418 concentration baseline of 300 μg / ml was applied to PBLs transduced on the first 7 to 9 days.

After this initial period, G418 concentration was adjusted to the value determined in the toxic dose analysis.

For the 10 donors tested, G418 toxicity doses ranged from 300-500 μg / ml when the first 10 ⁵ cell concentration was used.

After transduction and selection, PBLs were incubated for one week in fresh medium without G418.

This stage of recovery is important to enhance cell viability and increase cell number for subsequent HIV-1 attack assays (about 3-5 times greater than with G418).

Expression of Transgene in Transduced PBLs. Expression of ribozyme, antisense or TAR sequences in transduced PBLs was assessed.

Figures 16A-16C show representative aspects of the Northern analysis.

In RRz2 and LNL6 transduced cells (FIG. 16A), both spliced and unspliced transcripts (3.2 kb and 2.4 kb) containing neo ^r -ribozyme or neo ^r messages are neo ^r Detected using specific probes.

The predominant RNA species was the unconjugated transcript.

When blots were hybridized with ribozyme specific probes, the same behavior was observed for RRz2 RNA only.

In RASH5 transduced PBLs, two transcripts (spliced and unspliced) from 5 'LTR were detected using a 550 bp probe and confirmed the predicted size (4.8 kb and 4.0 kb) (Figure 1B).

However, the short transcript expected from the internal CMT promoter was not expressed, indicating that the CMV promoter was removed from this construct.

The R20TAR vector generated 4.6 kb transcript from the 5 'LTR hybridizing to the TAR probe as expected (Figure 16C), due to inactivation of the splice donor in the LXSN.

Inhibition of HIV-1 Replication in Human PBLs.

To analyze the relevance of the PBL assay system for the study of HIV-1 gene therapy, HIV challenge experiments were conducted on transduced PBLs using both laboratory strain (IIIB) and primary clinical isolate (82H). It was.

Infection was performed twice and repeated for 3-5 batches of PBLs.

HIV replication was recorded at various time points by measuring the p24 antigen level of the culture supernatant.

In challenge experiments with HIV-1 IIIB strains, PBLs expressing ribozyme (FIG. 17A), antisense (FIG. 17B) and TAR decoy (FIG. 17C) were applied to PBLs transduced with the corresponding control vector. In comparison, p24 production was significantly reduced (70%).

A relatively low degree of inhibition (40% to 70%) was also observed in transduced but not selected PBLs.

We also evaluated the resistance to infection of primary HIV-1 isolates against transduced and selected PBLs. Primary clinical isolates 82H can be obtained directly from PBMCs of patients and are characterized as T cell tropic and synthium-induced isolates.

For IIIB, 82H replication (analyzed by p24 anti-win ELISA) was inhibited to a similar extent as in HIV-1 IIIB directed PBLs (Figures 18A-18C).

These results indicate that these transgenes carried into human PBLs via retroviral vectors can inhibit HIV-1 replication in primary hematopoietic cells.

Analysis of Transduced PBLs.

To investigate the potential effects of transduction and construct expression on PBL proliferation, a [ ³ H] -thymidine-capture assay was performed on all transduced selected PBLs.

Compared to normal non-transduced PBLs, there was no noticeable depressive effect on transgene contructs and vector transduced PBLs (p <0.02) (FIG. 19).

Moreover, FACS analysis demonstrates that CD4 surface markers remain unchanged after transduction (Table 5).

This suggests that the inhibitory effects observed in HIV-1 challenge assays are not due to a decrease in the number of CD4 receptors on transduced PBLs.

Table 5. CD4 / CD8 Surface Markers in PBLs by FACS Analysis

Total PBLs = total non-transduced total PBLs; CD4 ⁺ PBLs = non-transduced CD4 ⁺ enhanced PBLs; LNL6-PBLs = CD4 ⁺ enhanced PBLs transduced with LNL6 virus; RRz2-PBLs = CD4 ⁺ enhanced PBLs transduced with RRz2 virus.

20 shows the results of CEM T4 T-lymph cell lines transduced by virus and subjected to G418 selection.

The entire population includes cells attacked with HIV-1 with random integrants and various product expression levels.

21A-21B show the results of complex ribozymes directed at tat, RzM and ribozymes directed at both the packaging site and tat, RRzpsi / M.

Review

Ultimately, for gene therapy used to inhibit in vivo HIV-1 replication, such gene therapy approaches must first be tested in a laboratory system.

Today, these experimental systems relate primarily to human T lymphocyte lines and there are no published data available for primary PBLs (Yu, M. et al. 1994).

To obtain preclinical data, it is important to test anti-HIV-1 gene therapy in primary hematopoietic cells.

These cells are the main targets for HIV-1 infection and replication, and the cell lines and PBLs have very different response and growth characteristics in vitro manipulation and responsiveness to HIV-1 infection.

This is the CD4 ⁺ PBL population of HIV-1 gene therapy.

For this reason, we conducted this study to establish a PBL analysis system.

In this study, three different retroviral vectors expressing ribozyme, antisense or TAR decoy genes were tested for anti-viral efficacy in human PBLs.

Although they were constructed in other retroviral vectors, they were shown to be similarly effective in HIV-1 protection assays without apparent cytotoxicity, as measured by ³ H-thymidine binding assays and FACS assays.

This observation shows the possibility of PBLs as a target for HIV-1 gene therapy.

Several points should be pointed out when using PBLs as analytical systems or as therapeutic target cells.

First, high viral titers are critical to obtaining efficient transduction of PBLs.

This is especially the case when transduced PBLs are undesirable for screening with G418.

We tested both unselected and selected as transduced with high titers of therapeutic retrovirus (> 10 ⁶ cfu / ml).

Although lower than those observed in selected PBLs, these non-selected PBLs were also found to be resistant to HIV-1 infection, which was clinically demonstrated for ex vivo transduced PBLs without G418 screening. It can be used as.

However, G418 screening allows the use of low titers of viruses for in vitro testing of gene therapy.

We can develop a two-step G418 sorting process and thereby easily perform complete sorting.

This process contributes to minimizing the time of incubation in vitro and thereby reducing any modifications to the T cell population.

In this experiment, two-week incubation of in vitro PBLs had no significant effect on surface markers (CD4 ⁺ and CD8 ⁺ ).

This period (2 weeks) will be sufficient for ex vivo manipulation of PBLs for therapeutic purposes.

Retroviral vector design is another important aspect for efficient gene transfer and expression.

There is no direct comparison of the three vector designs used in this study, but two observations are noteworthy.

First, all transgenes regulated by viral LTR (but not from the CMV internal promoter for one construct) are efficiently expressed in a constitutive manner in human primary hematopoietic cells.

Second, the strategy of inserting ribozymes into 3 'untranslated regions of genes such as neo ^r of retroviral vectors appears to be as efficient in PBLs as in T cell lines.

This finding will be useful for future improvements in gene therapy design.

In conclusion, the transduction of human primary PBLs and their protection in vitro from HIV-1 may be achieved using the experimental methods described herein.

This not only provides a useful system for the evaluation of gene therapy in vitro, but also forms the basis for HIV-1 gene therapy targeted to CD4 ⁺ lymphocytes.

Table 6. Animal Retroviruses

AIDS-related Virus (ARV)

Avian Erythroblastosis Virus

Avian Keukosis Virus (ie Lympoid Leukosis Virus)

Avian Myeloblastosis Virus

Avian Reticuloendotheliosis Virus

Avian Sarcoma Virus

Baboon Endogenous Virus

Bovine Leukemia Virus

Bovine Lentivirus

Bovine Syncytial Virus

Goat encephalitis-arthritis virus (i.e., coat leukoencephalitis virus)

Avian Melocytomatosis Virus

Corn Snake Retrovirus

Chicken Syncytial Virus

Duck Infectious Anemia Virus

Deer Kidney Virus

Equine Dermal Fibrosarcoma Virus

Equine Infectious Anemia Virus

Esh Sarcoma Virus

Feline Immundeficiency Virus

Feline Leukemia Virus

Feline Sarcoma Virus

Feline Syncytium-forming Virus

Fujinami Sarcoma Virus

Gibbon Ape Lekemia Virus (ie, Simian Lymphoma Virus or Simian Myelogeneous Leukemia Virus)

Golden Pheasant Virus

Human Immunodeficiency Virus 1 (HIV-1)

Human Immunodeficiency Virus 2 (HIV-2)

Human T-Lymphotropic Virus 1: HTLV-1

Human T-Lymphotropic Virus 2: HTLV-2

Human T-Lymphotropic Virus 3: HTLV-3

Lymphoproliferative Disease Virus

Myeloblastosis-associated Virus

Myelocytomatosis Virus

Mink Cell Focus-Inducing Virus

Myelocytomatosis Virus 13

Mink Leukemia Virus

Mouse Mammary Tumor Virus

Mason-Pfizer Monkey Virus

Murine Sarcoma Virus

Myeloid Leukemia Virus

Progressive Pneumonia Virus

Mouse Leukemia Virus

Rat Sarcoma Virus

Rous-Associated Virus 0

Rous-Associated Virus 60

Rous-Associated Virus 61

Reticuloendotheliosis-Associated Virus

Reticuloendotheliosis Virus

Reticuloendotheliosis Virus-Transforming

Ring-Necked Pheasant Virus

Rous Sarcoma Virus

Monkey Foamy Virus

Simian Immunodeficiency Virus

Spleen Focus-Forming Virus

Squirrel Monkey Retrovirus

Spleen Necrosis Virus

Sheep Pulmonary Adenomatosis / Carcinoma Virus

Simian Sarcoma-Associated Virus (Wooly monkey Leukemia Virus)

Simian Sarcoma Virus (Wooly Monkey Virus)

Table 7. Packaging Sequence List:

Reticuloendotheliosis Virus (Rev)

Genome : Wilhelmsen, et al. J. Virol. 52: 172-182 (1984).

Bases 1-3149: Shimotohno, et al. Nature 285: 550-554 (1980).

Base 3150-3607 Packaging Sequence (Ψ): 144-base between Kpn I site at 0.676 kbp and 0.820 kbp for 51 termini of the provirus

J. Embretson and H. Temin, J. Virol. 61 (9): 2675-2683 (1987)

2. Human Immunodeficiency Virus type 1: HIV-1

Genome : Gallo et al. Science 224: 500-503 (1984)

Packaging Sequence (Ψ): 19 base pairs between 5 ′ LTR and gag gene initiation codon.

A. Lever, J. Virol. 63 (9) 4085-4087 (1989).

3. Moroni Mouse Leukemia Virus (Mo-MLV)

Genome : Shinnick, et al. Nature 293: 543-548 (1981).

Packaging sequence (Ψ): 350 nucleotides between the splice site and the AUG site encoding a gag protein sequence.

Secondary packaging sequence (Ψ +): only at 5 'half of U5 site. J. Murphy and S. Goff, J. Vlrol. 63 (1): 319-327 (1989).

4. Avian Sarcoma Virus (ASV)

Genome : Neckameyer and Wang, J. Virol. 53: 879-884 (1985).

Packaging Sequence (Ψ): 150 base pairs between 300-600 bases from the left (gag-pol) end of the provirus. P. Shank and M. Linial, J. Virol. 36 (2): 450-456 (1980).

5. Raus Sarcoma Virus (RSV)

Genome : Schwarts et al., Cell 32: 853-869 (1983).

Packaging Sequence (Ψ): 230 base pairs from 120-base (beginning with PB site) to 22-base before gag start codon. S. Kawai and T. Koyama (1984), J. Virol. 51: 147-153.

6. Bovine Leukosis Virus (BLV)

Genome : Couez, et al. J. Virol. 49: 615-620 (1984),

Base 1-341; Rice, et al., Virology 142: 357-377 (1985),

Base 1-4680; Sagata, et al., Proc. Natl. Acad. Sci. 82: 677-681 (1985),

Complete BLV provirus,

Packaging Sequence (Ψ): Predicted by the inventors between the end of the primer binding site at about base 340 and the initiation codon for gag at about bases 41-8.

Claims (50)

An oligonucleotide compound comprising a ribozyme targeted to a GUA target site at nucleotide 5849 of an HIV-1 SF-2 isolate, wherein the ribozyme has the following structure: Wherein each X is the same or different and represents a nucleotide whose sugar, phosphate or base may be modified or substituted; Each of A, C, U and G represents a ribonucleotide in which its sugar, phosphate or base may be unmodified, modified or substituted; 3'- AAG ... AGUCX-5 'defines a conserved catalyst region; Each of (X) _n A and (X) _{n '} defines a nucleotide of a sequence capable of hybridizing with a given sequence in an HIV-1 SF-2 isolate; Each ★ represents a base pair between nucleotides located on either side of it; Each solid line represents a chemical bond that provides a covalent bond between nucleotides located on either side thereof; a represents an integer defining the number of nucleotides, provided that a can be 0 or 1 and if 0 _A at 5 'of (X) _a is bonded to X at 3' of (X) _a ; m and m 'each represent an integer equal to or greater than 1; (X) _b represents an oligonucleotide, b represents an integer equal to or greater than 2), or The ribozyme has the following structure: Wherein each X is the same or different and represents a nucleotide whose sugar, phosphate or base may be modified or substituted; Each of A, C, U and G represents a ribonucleotide in which its sugar, phosphate or base may be unmodified, modified or substituted; 3'- AAG ... AGUCX-5 'defines a conserved catalyst region; Each of (X) _n A and (X) _{n '} defines a nucleotide of a sequence capable of hybridizing with a given sequence in an HIV-1 SF-2 isolate; m represents an integer from 2 to 20; None of the nucleotides (X) _m form Watson-Crick base pairing with other nucleotides in the compounds; Each syncope represents a chemical bond that provides a covalent bond between nucleotides located on either side of it) Has or The ribozyme has the following structure: (Wherein 3 '(X) _P4 ... (X) _P1-5 ' defines the storage catalyst region, Each of (X) _F4 and (X) _F3 defines a nucleotide of a sequence capable of hybridizing with a predetermined sequence in an HIV-1 SF-2 isolate; Each solid line represents a chemical bond that provides a covalent bond between nucleotides located on both sides thereof; F3 represents an integer defining the number of nucleotides in an oligonucleotide, provided that F3 is equal to or greater than 3; F4 represents an integer defining the number of nucleotides in the oligonucleotide, provided that F4 is 3 to 5; Each of (X) _P1 and (X) _P4 represents an oligonucleotide having a predetermined sequence such that (X) _P4 forms a base pair with 3-6 bases of (X) _P1 ; P1 represents an integer defining the number of nucleotides in the oligonucleotide, except that P1 is 3 to 6 and the sum of P1 and F4 is 9; (X) _P2 and (X) _P3 each of (X) _P2 is (X) oligonucleotide having a predetermined sequence to form at least three nucleotide base pairs of the _P3 represents an oligonucleotide; Each dotted line independently represents a chemical bond that provides a covalent bond between nucleotides located on both sides thereof or indicates the absence of such a chemical bond; (X) _L2 represents an oligonucleotide that may or may not be present, provided that ( ₂ ) _L2 represents an integer equal to or greater than 3 when (X) _L2 is present) Oligonucleotide compound having a. The oligonucleotide compound of claim 1, wherein the ribozyme is hemmerhead ribozyme. The oligonucleotide compound of claim 1, which is a single ribozyme. The oligonucleotide compound of claim 1, wherein the oligonucleotide is a plurality of labozymes targeted to other sites in the tat . The oligonucleotide compound of claim 1 comprising a continuous nucleotide encoded by the sequence: Where T, G, A and C each represent a nucleotide. The oligonucleotide compound of claim 1 comprising a continuous nucleotide encoded by the sequence: Where T, G, A and C each represent a nucleotide.  The oligonucleotide compound of claim 1, further comprising a continuous nucleotide encoded by the sequence: Where T, G, A and C each represent a nucleotide. The oligonucleotide compound of claim 4, wherein the plurality of target sites in tat are at 5792, 5886, or 6042 nucleotides of the HIV-1 SF-2 isolate. A transfer vector comprising RNA or DNA, or a combination of DNA and RNA, that produces the compound of claim 1 upon transcription. 10. The transfer vector according to claim 9, which contains a eukaryotic promoter for expression of said compound in eukaryotic cell types that can be infected by HIV. An exo vivo cell comprising an oligonucleotide, the oligonucleotide comprising a nucleotide having a sequence which, upon transcription, produces a compound comprising a ribozyme targeted to a CUA target site at nucleotide 5849 of an HIV-1 SF-2 isolate To exo vivo cells. The cell of claim 11, wherein said compound is the compound of claim 1. 13. The cell of claim 12, wherein said ribozyme can inhibit HIV-1 replication in human primary hematopoietic cells. The cell of claim 11, wherein the nucleotide sequence is part of a transfer vector structure. The cell of claim 14, wherein the transfer vector is an adenovirus associated transfer vector, Mo-MLV, or an amphoteric retroviral vector. The cell of claim 14, wherein the transfer vector is a retroviral vector. 17. The cell of claim 16 wherein the nucleotide sequence is in the 3 'untranslated region of the neomycin resistance gene of the retroviral transfer vector. The cell of claim 17 wherein the transfer vector is derived from an LNL6 vector. The cell of claim 11, wherein the oligonucleotide comprises a nucleotide having the sequence: Where T, G, A and C each represent a nucleotide. The cell of claim 11, wherein the oligonucleotide comprises a nucleotide having the sequence: Where T, G, A and C each represent a nucleotide. The cell of claim 11, wherein the oligonucleotide comprises a nucleotide having the sequence: Wherein each of T, G, A and C represents nucleotides. 22. The method according to any one of claims 11 to 13 and 15 to 21, wherein the cells are hematopoietic stem cells, progenitor cells, delegated progenitor cells, T lymphocyte progenitor cells, immature T lymphocytes, mature T lymphocytes, bone marrow progenitor cells. Or cells that are monocytes / macrophage cells. The cell of claim 22, wherein the cell is a progenitor cell. The cell of claim 22, wherein the cell is T lymphocyte. The cell of claim 22, wherein the cell is a human primary hematopoietic cell. A pharmaceutical composition for making human cells resistant to HIV infection containing a compound of any one of claims 1 to 8 or a transfer vector according to claim 9 or 10 in a pharmaceutically acceptable carrier. The cell of claim 14, wherein the cell is a hepatic stem cell, progenitor cell, delegated progenitor cell, T lymphocyte progenitor cell, immature T lymphocyte, mature T lymphocyte, myeloid progenitor cell or monocyte / macrophage cell. Oligonucleotide compounds comprising a continuous nucleotide having the sequence or encoded by a continuous nucleotide having the sequence (Wherein T, G, A and C each represent a nucleotide targeted to a GUA target site at nucleotide 5849 of the HIV-1 SF-2 isolate). The oligonucleotide compound of claim 28 which is a single ribozyme. The oligonucleotide compound of claim 28, wherein the oligonucleotide is a plurality of ribozymes targeted to other sites in the tat . The oligonucleotide compound of claim 28, comprising a continuous nucleotide having the sequence or encoded by the continuous nucleotide having the sequence Where T, G, A and C each represent a nucleotide. The oligonucleotide compound of claim 28, further comprising a contiguous nucleotide having the sequence or encoded by a contiguous nucleotide having the sequence Where T, G, A and C each represent a nucleotide. The oligonucleotide combinations of claim 30, wherein the plurality of target sites in tat are at 57922, 5886, or 6042 nucleotides of an HIV-1 SF-2 isolate. A transfer vector comprising RNA or DNA, or a combination of DNA and RNA, that produces the compound of claim 28 upon death. 35. The transfer vector of claim 34 containing a eukaryotic promoter for expression of said compound in a eukaryotic cell type that can be infected by HIV. Exo vivo cells comprising the oligonucleotide compound of claim 28. The cell of claim 36, wherein the compound is capable of inhibiting HIV-1 replication in human primary hematopoietic cells. 38. The cell of claim 36 or 37 wherein said nucleotide sequence is part of a transfer vector structure. The cell of claim 38, wherein the transfer vector is an adenovirus associated transfer vector, Mo-MLV, or an amphoteric retroviral vector. The cell of claim 38, wherein the transfer vector is a retroviral vector. 41. The cell of claim 40, wherein the nucleotide sequence is at the 3 'untranslated region of the neomycin resistance gene of the retroviral transfer vector. The cell of claim 41 wherein the transfer vector is derived from an LNL6 vector. The cell of claim 36, wherein the oligonucleotide has the following sequence or comprises consecutive nucleotides encoded by: Where T, G, A and C each represent a nucleotide. The cell of claim 36, wherein the oligonucleotide further comprises consecutive nucleotides having or encoded by the following sequence. Where T, G, A and C each represent a nucleotide. 45. The method of any one of claims 36, 37 and 39-44, wherein the cells are hematopoietic stem cells, progenitor cells, delegate progenitor cells, T lymphocyte progenitor cells, immature T lymphocytes, mature T lymphocytes, bone marrow progenitor cells. Or cells that are monocytes / macrophage cells. 46. The cell of claim 45 wherein the cell is a progenitor cell. 46. The cell of claim 45 wherein the cell is T lymphocyte. 46. The cell of claim 45 wherein the cell is a human primary hematopoietic cell. A pharmaceutical composition for making human cells resistant to HIV infection containing a compound of any one of claims 28-33 or a transfer vector according to claim 34 or 35 in a pharmaceutically acceptable carrier. The cell of claim 38, wherein the cell is a hematopoietic stem cell, progenitor cell, delegated progenitor cell, T lymphocyte progenitor cell, immature T lymphocyte, mature T lymphocyte, myeloid progenitor cell or monocyte / macrophage cell.