WO1999013070A1

WO1999013070A1 - t-RNA PRIMER, PRODUCTION AND USE FOR INHIBITING REVERSE TRANSCRIPTASE

Info

Publication number: WO1999013070A1
Application number: PCT/EP1998/005778
Authority: WO
Inventors: Horst Domdey; Hans Joachim Gross; Stefan Schäfer; Hermann Heumann
Original assignee: Multigene Biotech Gmbh
Priority date: 1997-09-11
Filing date: 1998-09-10
Publication date: 1999-03-18
Also published as: DE19739956C1; AU9539898A

Abstract

The invention relates to a nucleic acid construction characterized in that the nucleic acid derived from a t-RNA existing in nature is extended at the 3' terminus. Extended 3' terminus is not complementary, or only partly, to the nucleotides to be found upstream from a primer binding site. The invention also relates to its production and use for inhibiting reverse transcriptase.

Description

tRNA primer, its preparation and use for inhibiting the

Reverse transcriptase

The present invention relates to a nucleic acid construct in which the nucleic acid derived from a naturally occurring tRNA is extended at the 3 'end and the extended 3' end is not or only partially complementary to the upstream nucleotides of a PBS region, and its manufacture and use for the inhibition of reverse transcriptase.

Transfer RNA (tRNA) molecules are ubiquitous in every cell. Common to all of them is their function as an adapter molecule in protein biosynthesis. During translation, they translate the triplet code of the mRNA into the colinear protein sequence through the specific codon-anticodon interactions. With regard to this original interaction with the ribosomal complex, they have a uniform structure that has been preserved evolutionarily (FIG. 1).

The transcription of the tRNA genes takes place as a larger precursor molecule, which is processed into the mature tRNA by nucleus-localized enzymes. The processing of the precursors includes the removal of the 3 'and 5' flanking sequences as well as the splicing out of an intron which may be present.

The function of the tRNA is not limited to protein synthesis. In addition to regulating the transcriptions of some aminoacyl tRNA Synthetase genes or the involvement of tRNA in chlorophyll biosynthesis, it plays a fundamental role in the replication of retroviruses.

The enzyme involved in the replication of retroviruses, reverse transcriptase, is, like DNA-dependent DNA polymerases, unable to start replicative DNA synthesis de novo. In the reverse transcription of the viral RNA into DNA, a tRNA acts as a primer (tRNA primer), which is recruited from the host's cytoplasmic tRNA pool during the assembly of a new virus particle. Studies have shown that in the Rous sarcoma virus, reverse transcription takes place using the tRNA ^{I rp} as a primer.

The occurrence of reverse transcriptase is not limited to retroviruses alone. So far, it has been detected in hepadnaviruses, class II plasmids and in mobile genetic elements such as retrotransposons, retrons and class 1 introns. For the retrovirus human immunodeficiency virus type 1 (HIV 1) it could be shown that the replication takes place with the host's own tRNA ^{LY '} (FIG. 2) as a tRNA primer.

The viral RNA of HIV 1 is 9.7 kb in size and has the same genome organization as other retroviruses (see FIG. 3). Gag codes for matrix proteins, pol for reverse transcriptase, integrase and protease and env for the precursor protein gpl60, which is cleaved into gpl20 and gp41. Characteristic are the LTR regions flanking the genome, which are essential for virus replication. The primer binding site (PBS) is directly adjacent to the U5 region and comprises 18 nucleotides that are complementary to the 3-end of the tRNA ^YS ₃ .

The first steps in the retroviral life cycle are binding and Fusion with the host cell. The adsorption takes place through an interaction of the gpl20 protein with the CD4 molecule of a suitable host cell. The merger is believed to be mediated by gp41. After penetration and release of the ribonucleocapsid into the cytoplasm, the viral RNA is replicated by the reverse transcriptase. The tRNA primer is specifically selected by the reverse transcriptase or its non-processed Gag-Pol precursor protein and is characteristic of a retrovirus strain. After partial unwinding of the 3 ^' terminus, the tRNA primer binds to the PBS region of the viral RNA and initiates the (-) strand DNA synthesis by means of reverse trariscriptase at the 3' - terminal adenosine residue, the (-) DNA Strand is covalently linked to the tRNA ^LyS ₃ primer (FIG. 4). The synthesis of the DNA is accompanied by a degradation of the RNA template by the RNase H activity of the reverse transcriptase. The (-) strand-stop DNA performs an inter- or intramolecular template change mediated by the reverse transcriptase and the nucleocapsid protein NCp7. binds to the R region of the 3-LTR and the (-) strand DNA synthesis continues. The template is simultaneously degraded by the RNase H, with the exception of a sequence of polypurins, which serves as a primer in the subsequent synthesis of the (+) strand. The PBS is removed by the RNase H * activity and the tRNA serves as a template in completing the (+) strand-stop DNA synthesis. The reverse transcription of the tRNA template stops at the first modified nucleotide of the tRNA ^Lys ₃ . After degradation of the tRNA down to the 3 ^' -terminal adenosine, a second template change is carried out and the synthesis of the (+) strand DNA is completed. The remaining adenosine residue can only be detected in the circular stage and not in the integrated provirus.

The tRNA ^Lys _{3 is selected} as the primer of the (-) strand synthesis during virus assembly from the tRNA population of the host cell.

The encapsulation of the tRNA is independent of the presence of viral RNA.

The object of the present invention is to find compounds which inhibit the process of reverse transcription.

It has now surprisingly been found that a processing-deficient tRNA primer with a non-complementary one

3-Terminus to the viral RNA the cDNA synthesis of a homologous HIV-1

RNA templates efficiently inhibited. In contrast, tRNA primers were efficiently elongated with a 3 ^' flank that was completely complementary to the viral RNA. When Avian Myeloblastosis Virus (AMV) reverse transcriptase was used in a non-homologous system, an elongation deficiency of the tRNA primers with a non-complementary 3 'terminus was also observed. There is thus a general principle for the inhibition of cDNA synthesis by a tRNA primer which is partially or completely non-complementary at its 3 ^' terminus.

The invention therefore relates to a nucleic acid construct in which the nucleic acid derived from a naturally occurring tRNA is extended at the 3 'end and the extended 3' end is not or only partially complementary to the upstream nucleotides of a PBS region . The naturally occurring tRNA usually comes from a mammal, especially from humans. In the case of an only partially complementarily extended 3 'end, the extended tRNA at the 3' end of the extension is not complementary to the sequence or viral partial sequence adjacent to the PBS region. The extension is usually made directly to the CCA terminus. Generally this is 3 'end by at least approx. 1-500, preferably by at least approx. 3-

20, in particular by at least approx. 4- 1 8, especially by at least approx. 14-

1 8 nucleotides extended. The general principle of the present

The invention is summarized schematically in FIG. 5.

According to the present invention, the nucleic acid construct can be both in the form of a gene, in particular in the form of a pseudogen (see below), as well as an RNA in the form of a tRNA.

A particularly preferred object is a nucleic acid construct according to the invention, in which the nucleic acid derived from a naturally occurring tRNA is additionally mutated. Such constructs are also referred to as pseudogenes, since the pseudogenes cannot express mature tRNA, but only precursors of tRNAs. Such tRNA pseudogen products can be expressed in vivo and have sufficient stability. In connection with the present invention, it has also been shown that the nucleic acid construct according to the invention in the form of a pseudogen made it possible to express a stable, non-processable precursor tRNA primer, the 3 ^' flanking sequence of which did not occur during the maturation of the tRNA precursor was degraded. The particularly preferred nucleic acid construct is a particularly efficient inhibitor of the initiation of, for example, viral replication.

According to the present invention, the mutation is usually in the acceptor strain, D strain, anticodon strain and / or T strain of the nucleic acid derived from a naturally occurring tRNA (see FIG. 1), preferably in the T strain and / or D strain, in particular in the T strain. Mutations in the range of nucleotides 22-25 and / or 49-52 are preferred, preferably in the range of nucleotides 49-52. The mutation is usually a substitution, especially a substitution of the nucleotides A23 -> T, C25-> G, A50-> C, G5 1 -> A or G52-> C, especially A50-> C, G51 -> A or G52-> C, especially A50—> C and G52—> C or G51 -> A. In addition, the mutation can also be one or more insertions or deletions.

The following table shows an exemplary compilation of pseudogens according to the invention whose expression products (here: tRNA ^vs primer) are not or only partially processed.

Tab. 1

The processing deficiency of the nucleic acid constructs according to the invention can lie in a disruption of the secondary and / or tertiary structure, as well as in a partial unwinding of the T strain while maintaining the interaction between the D and T loops, as was observed with K240. In K238, the T strain is partially unfolded and the interaction between G 19 and C56 could no longer be demonstrated, which indicates a no longer intact "L" structure. In general, local helix removal by at least one base mismatch is opposed to a processing deficiency the 3 ^' tR noses are sufficient.

Another preferred embodiment of the present invention relates to nucleic acid constructs which are additionally derivatized. The derivatization achieves further stability with respect to nucleases, such as RNases or DNases, which are present in particular in body fluids. Examples of derivatized nucleic acids are nucleic acids with modified internucleotide phosphate residues, such as methylphosphonates, phosphorothioates or phosphorodithioates, phosphoramides or phosphate esters, in particular phosphorothioate derivatives (Eckstein (1985) Annu. Rev. Biochem. 54, 367-402) or PNA derivatives ( Hyrup and Nielsen (1996) Bioorg. Med. Chem. 4, 5-23). Further examples of derivatizations can be found in Uhlmann E. & Peyman, A. (1990) Chemical Reviews 90, 543-584, No. 4th The nucleic acid construct according to the invention can be genetically engineered, for example, by expression of the corresponding transgene, e.g. B. in an SP6 or T7 system (Milligan & Uhlbeck (1989) 1 80, 5 1 -62), enzymatically or chemically, for example by the phosphoramidite method (Caruthers et al. (1987) Methods Enzymol. 154, 287- 313). The construct is then optionally derivatized, isolated and / or purified by methods known to those skilled in the art.

Another object of the present invention therefore relates to a method for producing the nucleic acid construct according to the invention, in which (a) the nucleic acid construct according to the invention is genetically, enzymatically or chemically synthesized and then optionally derivatized (b), (c) isolated and / or (d) is cleaned.

The nucleic acid construct according to the invention is preferably suitable as an inhibitor of reverse transcriptase in, for example, retroviruses, hepadnaviruses, class II plasmids or mobile genetic elements such as retrotransposons, retrons or class I introns. The nucleic acid construct according to the invention is particularly suitable as an inhibitor of the initiation of the reverse transcription of RNA into DNA in retroviruses, such as, for. B. with Onco, Lenti or Spuma viruses, especially with the human T-cell lymphotropic virus type 1 or 2 (HTLV-1, HTLV-2) and with the human immunodeficiency virus type 0, type 1 or type 2 (HIV-0 , HIV-1, HIV-2), but also in the case of RNA tumor viruses. The nucleic acid construct according to the invention is therefore preferably derived from virus-specific host tRNAs.

Particularly preferred is the inhibition of reverse transcription in HIV 1 by means of a nucleic acid construct according to the invention which is derived from the naturally occurring tRNA ₃ , since all have so far known HIV 1 isolates, the PBS region is highly conserved and is complementary to the tRNA ^LYS ₃ .

Another subject therefore relates to the use of the nucleic acid constructs according to the invention for inhibiting reverse transcriptase.

A further possibility of inhibiting the initiation of the reverse transcription is a deletion of the gene for the tRNA primer or a substantial part thereof, so-called “knock-outX, the use of a so-called antisense nucleic acid directed against the viral RNA or DNA, for example an antisense RNA, or one or more mutageneses in the regions of viral RNA or DNA which are important for initiation (see, for example, Betsy, TK et al. (1998) Nature Medicine, 4, 285-290).

Therapeutic or preventive use in humans, e.g. B. as a continuous infusion, is generally in the form of a medicament, which, if appropriate, further additives and auxiliaries, such as. B. contains an isotonic saline solution, calcium phosphate, nuclease inhibitors and / or stabilizers. The nucleic acid construct according to the invention can also be present in the drug in the form of a peptide / tRNA complex (Morris et al. (1997) Nucleic Acids Res. 25, 2730-2736).

It can also be used in humans as part of gene therapy. For this purpose, the nucleic acid construct according to the invention is combined, for example, with a virus vector and / or liposomes as additives. Suitable viral vectors are, for example, adenovirus vectors, preferably replication-deficient adenovirus vectors (see, for example, WO95 / 00655) or adeno-associated virus vectors, especially adeno-associated virus vectors which consist exclusively of two inverted terminal repeat sequences (ITR) exist (see e.g. Chiorini, JA et al. (1995) Human Gene Therapy 6, 1531-1541). For lipofection of cells, e.g. B. of T cells, for example, lipid mixtures are suitable as in Feigner et al. (1987) Proc. Natl. Acad. Be. USA 84, 7413-7417. Lipofection is particularly suitable when the nucleic acid constructs according to the invention in the form of their tRNAs directly in the target cells, e.g. B. HIV-infected T cells to be spent.

In the gene therapy use of the corresponding pseudogen constructs according to the invention in the form of z. B. viral vectors, the pseudogen constructs according to the invention are expressed in vivo. The expression products (tRNA primers) can thus inhibit retroviral DNA synthesis on site, for example.

The present invention therefore furthermore relates to a medicament comprising a nucleic acid construct according to the invention and, if appropriate, suitable additives and auxiliaries, and the use of a nucleic acid construct according to the invention for producing a medicament for the therapy or prevention of a retroviral disease.

In general, the drug is used for the somatic gene therapy of retroviral diseases or for preventive protection against retroviral infections. For this purpose, bone marrow cells of the patient are, for example, in vitro using the nucleic acid construct mentioned, if appropriate in a suitable method

Expression vector transformed and then reinjected into the patient's bone marrow to thereby obtain virus-resistant T cells. DESCRIPTION OF THE FIGURES

1 shows a schematic representation of the secondary and tertiary structure of a tRNA. The left representation shows the so-called cloverleaf structure. Invariant nucleotides are labeled. The right representation shows a three-dimensional structural model.

2 shows the nucleotide sequence of the tRNA ^YS ₃ from bovine liver, including modifications. S means the modification mcm5s2U and R means ms2t6A. The nucleotide sequence of the bovine tRNA ^YS ₃ is identical to the nucleotide sequence of the human tRNA ^LYS ₃ .

3 shows the schematic representation of the proviral genome organization of HIV-1.

Fig. 4 shows a schematic representation of the reverse transcription. - means single-stranded RNA, means DNA with intermolecular

Template change and - means single-stranded DNA.

5A-C show schematic representations of primer-template complexes, FIG. 5A showing a primer-template complex of the natural, wild-type primer, FIG. 5B a primer-template complex with a partially non-complementary primer, and FIG 5C shows a primer-template complex with a completely non-complementary primer. (1) stands for tRNA, (2) for tRNA sequence including CCA-3 'end, (3) for viral RNA and (5) for mutation within the tRNA primer. 6A and 6B show schematic secondary structures of the primer

Template complexes from HIV-RNA 1 and K208 or K210 (FIG. 6A) as well

HIV-RNA1 and K238 and K240, respectively (Fig. 6B).

7 shows the nucleic acid sequence in the form of the cloverleaf structure of the tRNA pseudogenes pHtK208, pHtK210, pHtK238 and pHtK240, black nucleotides differing from the wild-type sequence (mutations). The underline indicates the termination signal of the transcription. T marks the starting point of the transcription.

Fig. 8 shows an autoradiogram of a formamide-containing polyacrylamide gel of the different reaction approaches of a reverse transcription of HIV-RNA1 / 2 in the presence of different tRNA ^Lys ₃ primers. Lys3 stands for the naturally occurring tRNA ^LyS ₃ primer as a positive control. EP means expected elongation product.

EXAMPLES

1. Production of a tRNA ^y V pseudogen which is partially complementary to HIV-RNA

The start sequences HI-HE5FL, HI-TMT9 (Tab. 2) and pHtK208 and pHtK210 were used as templates in the construction of the pseudogenes pHtK238 and pHtK240.

Tab. 2

H I-HE5FL 5 'GGGGTACCACCATGATTACGCCAAGCTTGGC

H IT T5B 5 ^' -TTTAAAAAAATCTCTAGCAGTGGCGCCCGAACAGGG H IT T9 5' -AAAAAAAATCTCTAGCAGTCTCTAGC pHtK208 and pHtK210 were determined by an oligonucleotide-controlled PCR

Mutagenesis with HI-HE5FL and HI-TMT5B (Tab. 2) as the start sequence and pHtK15 or pKtK85 (Tab. 1) as template.

pHtK15 codes for a synthetic tRNA ^Lys ₃ pseudogen that carries two substitutions (A50-> C and G52-> C). pHtK85 codes for a synthetic tRNA ^Lys ₃ pseudogen that carries a substitution (G5 1 -> A).

The PCR mutagenesis was carried out using a method from Sarkar & Sommer (Sarkar, G. & Sommer, S.S. (1990), BioTechniques 8, 404-407). After their phosphorylation, the PCR products were cloned into the Smal restriction site of pUC 19.

The 3'-flanking sequence of the constructs pHtK208 and pHtK210 is completely complementary to the nucleotides of the HIV-RNA consensus sequence following the PBS upstream (FIG. 6A). The constructs pHtK238 and pHtK240 are complementary only in the first 10 nucleotides of the 3 ^' flank of the tRNA gene (FIG. 6B). The following 10 bases are a duplication of the previous one and do not enter into any further base pairings with the viral RNA. The complete nucleic acid sequence of the constructs mentioned is shown in FIG. 7.

2. In tro-transcription of the tRNA ^ys ₃ pseudogenes

The plasmids pHtK208, pHtK210, pHtK238 and pHtK240 were in

HeLa nuclear extract transcribed under processing conditions (Arnold et al. (1986) Gene 44, 287-297). The plasmids pHtKWT2 and pHtKWT3 were used as the transcription standard corresponding mutations in the T strain of the tRNA are missing. The

Investigations were carried out with three different HeLa

Nuclear extract preparations (Dignam et al. (1983) Nucleic Acids Res. 11,

1475-1489; performed (Hl -K, H2-K and H3-K) to ensure that a possible processing deficiency is not only due to a variance in the extract used. The HeLa transcription was carried out in the presence of 4 mM magnesium chloride with the core extract H3-K for 70 min. The samples were analyzed on a denaturing 8% polyacrylamide gel.

The variation of the 3 ^' flanking sequence led to the desired result. The primary transcripts of constructs K210, K238 and K240 have a pronounced processing deficiency under the given reaction conditions. Less than 1% of the primary transcript was processed partially or up to mature tRNA, regardless of the core extract used. The proportion of processing products at K208 comprised an average of 4%. The efficiency of processing the wild-type pre-tRNA and thus the proportion of mature tRNA was, depending on the extract used, between 25% for H3-K and 80% for H 1 -K / H2-K. The yield of transcription product of the individual pseudogenes is comparable to one another, but varies depending on the HeLa nucleus extract used in relation to the wild-type construct. For the extract with the lowest processing activity H3-K a transcriptional yield of 95% of the wild type was determined, when using H1 -K / H2-K of approx. 65%.

3. Inhibition of initiation of reverse transcription

The following experiment shows the inhibition of reverse transcription by the tRNA pseudogen products as primers. As a template, the two T7 transcripts of the coding for a viral partial sequence

Plasmids pHIV-RNAl and pHIV-RNA2 used.

3.1 Construction of the plasmids pHIV-RNAl and pHIV-RNA2

The plasmid pHIV-RNAl was obtained by PCR with the oligonucleotides HIV-T7 and HIVRNA1-3 (Tab. 3). The DNA oligonucleotides used were partially complementary and acted as a start sequence and template for the thermostable polymerase, which filled in the non-double-stranded areas. The purified PCR product was phosphorylated and cloned into the Smαl restriction site of pUC19.

Starting from plasmid pHIV-RNAl, the plasmid pHIV-RNA2 was constructed by a megaprimer PCR mutagenesis (Sarkar & Sommer (1990), supra) with the start sequences HIV1-NEU, HIV2-NEU and HIV3-NEU (Tab. 3).

Tab. 11iv1-NEW 5 ^{'-TAATACGACΓACATATAGGTCIGGIAACIAGAG}

HIV2-NEW 5 ^' -CTGCTAGAGATTTTTACACCGTCTAGAGTGGTCTGAGG G

HIV3-NEW 5 -GGATGTCCCGGGCCCCTGTTACTTTCACTTTAAAGTCCCTG

HIV-RNA1 5'-CCACACTGACTAAAAGGGTCTGAGGGATCTCTAGTTAC

HIV-RNA2 5 ^{'HIV--CCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGA} RNA3 S'-GGATGACCCGGGCCCTTTCGCTTTCAAGTCCCTGTTCGG ACGCGCCΛCTG

HIV-T7 5'-TAATACGACTCACTATAGGTAACTAGAGATCCCTC

HIV-RNA1 corresponds to a consensus sequence of all previously published HIV 1 sequences (as of April 1996) and includes the PBS with 5'- and 3'-flanking sequences, for which an interaction with the tRNA ^ys ₃ during the initiation of reverse transcription has been demonstrated (Isel et al. (1995) J. Mol. Biol. 247, 236-250). HIV-RNA2 is identical to a partial sequence of the MAL isolate (Baudin et al. (1993) J. Mol. Biol. 229, 382-397;

Isel et al., (1996) EMBO 3.15, 917-924), but also only includes the sequence region interacting with the tRNA ^ys ₃ . Both the PBS and the upstream nine nucleotides are highly conserved and none of the known natural HIV isolates shows a variation in this area. The cluster of adenosine residues ("A" loop) following this sequence, for which an interaction with the anticodon region of the tRNA ^Lys ₃ has been demonstrated in vitro (Isel et al. (1993), supra), only varies in length between four (pHIV-RNAl) and five adenosine residues (pHIV-RNA2).

3.2 Reverse transcription with tRNA pseudogene products as primers

The transcripts of the recombinant plasmids pHt208 and pHtK210 as well as pHtK38 and pHtK240 obtained and isolated by an in vitro transcription with HeLa core extract were adjusted to a concentration of 100 nM with unlabelled T7 transcript and with a two-fold molar excess of HIV-RNA 1 resp Pre-hybridized HIV-RNA2. The

Hybrids were examined for their elongation in a reverse transcription with HIV-reverse transcriptase. The incubation period was between 10 and

30 min varies and the reaction mixture on a denaturing

Polyacrylamide gel analyzed. 5 '-labelled tRNA ^Lys _{3 was} used as a positive control, which was hybridized under the same conditions and reverse transcribed.

The test result documents that the completely complementary tRNA mutants K208 and K210 are elongated by the reverse transcriptase (FIG. 7). The efficiency of the elongation of K208 and K210 is identical (within the scope of the experimental fluctuation), but varies depending on the number of 3 'terminals Uridine residues of the HeLa transcript used and the one used

Template. The four to seven uridine residues of the 3 'flanking sequence resulting from the termination of the HeLa transcription are complementary or partially complementary to the immediately following sequence of four (HIV-RNA 1) or five (HIV-RNA2 ) Adenosine residues of the template RNA (Fig. 5A). The reverse transcriptase still tolerates the partial complementarity of the no longer interacting uridines, a maximum of three for HIVRNA1 and a maximum of two for HIV-RNA2. For K208 and K210, 85% of the elongation efficiency of the wild-type tRNA was determined when using HIV-RNA2 as a template, regardless of the number of terminal uridine residues. When using HIV-RNA 1 as a template and HeLa transcripts with up to six terminal uridine residues, the efficiency was comparable to that of HIV-RNA2 as a template. However, it decreased to 44% of the wild type in seven uridine residues. This result is also in line with previously published work. Reverse transcriptase from HIV has been shown to efficiently elongate RNA or DNA primers with up to three arbitrary mismatches at their 3 ^' end in vitro.

Reverse transcription using the tRNA ^ys ₃ mutants with only a partially complementary flank confirmed that the pseudogen transcripts K238 and K240 were not extended by the retroviral transcriptase, ie the partial complementarity of the 3 'flank of at least 14 nucleotides, depending on the number 3 ^' -terminal uridine residues are no longer tolerated by the enzyme (Fig. 6B).

Several test runs confirmed the result. SEQUENCE LOG

(1. GENERAL INFORMATION

(i) LOGIN

(A) NAME Dr Domdey, Horst

(B) ROAD Fasanenweg 6

(C) LOCATION Neuried

(D) LAND Germany

(E) POST LEITZAHL 82061

(n) DESCRIPTION OF THE INVENTION tRNA polymer, its production and use for the inhibition of reverse transcriptase

(in) NUMBER OF SEQUENCES 37

(IV) COMPUTER READABLE VERSION

(A) DISK Floppy disk

(B) COMPUTER IBM PC compatible

(C) OPERATING SYSTEM PC-DOS / MS-DOS

(D) SOFTWARE Patentin Release # 1 0, Version # 1 30 (EPA)

(v) Dates of the current registration application number DE 19739956 8

(2) INFORMATION ON SEQ ID NO 1

(l) SEQUENCE LABEL

(A) LONG 10 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(n) TYPE OF MOLECULE cDNA (m) HYPOTHETICAL YES (iv) ANTISENSE YES

(v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 1 AAGGGGAACC 10

(2) INFORMATION ON SEQ ID NO 2

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(π) TYPE OF MOLECULE cDNA (in) HYPOTHETICAL YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 2 AAGGGGCCAA CACCAAA 17

(2) INFORMATION ON SEQ ID NO 3

(i) SEQUENCE LABEL

(A) LONG 17 base pairs

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(n) TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 3 AAGGGGCCAA CACCAAA 17

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(n) TYPE OF MOLECULE cDNA (m) HYPOTHETICAL YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 4 AAGGGGAACC 10

(2) INFORMATION ON SEQ ID NO 5

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(u) TYPE OF MOLECULE (.DNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 5 AAGGGGCCAA CACCAAA 17

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(n) TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 6 AAGGGGCCAA CACCAAA 17

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(π) MOLECULE TYPE (.DNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 7 AAGGGGCCAA CACCAAA 17

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(n) TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 9 AAGGGGAACC 10

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(π) TYPE OF MOLECULE -DNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 10 AAGGGGCCAA CACCAAA 17

(2) INFORMATION ON SEQ ID NO 1 1

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(n) TYPE OF MOLECULE cDNA (in) HYPOTHETICAL YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 1 1 GGTAGGCAAT GATA 14

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(n) TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 12 CTGCTAGAGA 10

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(C) STRANDFORM single strand

(D) TOPOLOGY linear

(π) MOLECULE TYPE cDNA OH) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 16 CTGCTAGCGC TTCGGC 16

(2) INFORMATION ON SEQ ID NO 17

(i) SEQUENCE LABEL

(A) LONG 20 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(n) TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 17 CTGCTAGAGA CTGCTAGAGA 20

(2) INFORMATION ON SEQ ID NO 18

(0 SEQUENCE LABEL

(A) LONG 20 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(π) TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 18 CTGCTAGAGA CTGCTAGAGA 20

(2) INFORMATION ON SEQ ID NO 19

(0 SEQUENCE LABEL

(A) LONG 31 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

00 TYPE OF MOLECULE cDNA (in) HYPOTHETICAL YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 19 GGGGTACCAC CATGATTACG CCAAGCTTGG C 31

(2) INFORMATION ON SEQ ID NO 20

(0 SEQUENCE LABEL

(A) LONG 36 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

00 TYPE OF MOLECULE cDNA (in) HYPOTHETICAL YES (iv) ANTISENSE YES

(v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 20 TTTAAAAAAA TCTCTΛGCAG TGGCGCCCGA ACAGGG 36

(2) INFORMATION ON SEQ ID NO 21

(i) SEQUENCE LABEL

(A) LONG 26 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(π) TYPE OF MOLECULE cDNA (m) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 21 AAAAAAAATC TCTAGCAGTC TCTAGC 26

(2) INFORMATION ON SEQ ID NO 22

0) SEQUENCE IDENTIFIER

(A) LONG 33 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

00 TYPE OF MOLECULE cDNA (in) HYPOTHETICAL YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 22 TAATACGACT ACATATAGGT CTGGTAACTA GAG 33

(2) INFORMATION ON SEQ ID NO 23

(i) SEQUENCE LABEL

(A) LONG 39 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(π) TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES) ANTISENSE YES (\) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 23 CTGCTAGAGA TTTTTACλCC GTCTAGAGTG GTCTGAGGG 39

(2) INFORMATION ON SEQ ID NO 24

0) SEQUENCE LABEL

(A) LONG 41 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

00 TYPE OF MOLECULE cDNA (in) HYPOTHETICAL YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 24 GGATGTCCCG GGCCCCTGTT ACTTTCACTT TAAAGTCCCT G 41

(2) INFORMATION ON SEQ ID NO 25

0) SEQUENCE LABEL

(A) LONG 38 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

00 TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 25 CCACACTGAC TAAAAGGGTC TGAGGGATCT CTAGTTAC 38

(2) INFORMATION ON SEQ ID NO 26

(0 SEQUENCE LABEL

(A) LONG 41 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

00 TYPE OF MOLECULE cDNA (in) HYPOTHETICAL YES (\\) ANTISENSE YES (\) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 26 CCTTTTAGTC AGTGTGGAAA ATCTCTAGCA GTGGCGCCCG A 41

(2) INFORMATION ON SEQ ID NO 27

(0 SEQUENCE LABEL

(A) LONG 50 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(π) TYPE OF MOLECULE cDNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 27 GGATGACCCG GGCCCTTTCG CTTTCAAGTC CCTGTTCGGA CGCGCCACTG 50

(2) INFORMATION ON SEQ ID NO 28

(0 SEQUENCE LABEL

(A) LONG 35 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear 00 TYPE OF MOLECULE cDNA (in) HYPOTHETICAL YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 28 TAATACGACT CACTATAGGT AACTAGAGAT CCCTC 35

(2) INFORMATION ON SEQ ID NO 29

(0 SEQUENCE LABEL

(A) LONG 76 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(π) TYPE OF MOLECULE tRNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (\) TYPE OF FRAGMENT linear

(ix) FEATURE

(A) NAME'S KEY special feature

(B) LOCATION 1

(D) OTHER INFORMATION G is pG

(ix) FEATURE

(A) NAME'S KEY special feature

(B) LOCATION 6

(D) OTHER INFORMATION G is m ² G

(ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 10

(D) OTHER INFORMATION G is m ² G

(ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 27

(D) OTHER INFORMATION U is φ

0x) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 39

(D) OTHER INFORMATION U is φ

(ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 46

(D) OTHER INFORMATION G is m ⁷ G (ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 48

(D) OTHER INFORMATION C is m ⁵ C.

(ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 49

(D) OTHER INFORMATION C is m ⁵ C.

(ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 54

(D) OTHER INFORMATION T is Tm

(ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 55

(D) OTHER INFORMATION U is φ

(ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 58

(D) OTHER INFORMATION A is m'A

(ix) FEATURE

(A) NAME / KEY special feature

(B) LOCATION 76

(D) OTHER INFORMATION A is A _0H

(xi) SEQUENCE DESCRIPTION SEQ ID NO 29

GCCCGGAUAG CUCAGDCGGD AGAGCAUCAG ACUSUURAUC UGAGGGDCCA 50

GGGTUCAAGU CCCUGUUCGG GCGCCA 76

(2) INFORMATION ON SEQ ID NO 30

(0 SEQUENCE LABEL

(A) LONG 48 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

00 TYPE OF MOLECULE tRNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear

(xi) SEQUENCE DESCRIPTION SEQ ID NO: 30

GUGGAAAAUC UCUAGCAGUG GCGCCCGAAC AGGGACCUGA AAGCGAAG 48 (2) INFORMATION ON SEQ ID NO 31 30

(0 SEQUENCE LABEL

(A) LONG 36 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(u) TYPE OF MOLECULE tRNA (m) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 31 UUUUUUUAGA GAUCGUCACC GCGGGCUUGU CCCUGA 36

(2) INFORMATION ON SEQ ID NO 32

(0 SEQUENCE LABEL

(A) LONG 48 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

00 TYPE OF MOLECULE tRNA (in) HYPOTHETIC YES (iv) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 32 GUGGAAAAUC UCUAGCAGUG GCGCCCGAAC AGGGACCUGA AAGCGAAG 48

(2) INFORMATION ON SEQ ID NO 33

(0 SEQUENCE LABEL

(A) LONG 47 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(n) TYPE OF MOLECULE tRNA (m) HYPOTHETICAL YES 0v) ANTISENSE YES (v) TYPE OF FRAGMENT linear (xi) SEQUENCE DESCRIPTION SEQ ID NO 33 UUUUUUUU AG AGAUCGUCAG AGAUCGUCAC CGCGGGCUUG UCCCUGA 47

(2) INFORMATION ON SEQ ID NO 34

(0 SEQUENCE LABEL

(A) LONG 103 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(H) MOLECULE TYPE cDNA (in) HYPOTHETICAL YES

(iv) ANTISENSE YES

(v) TYPE OF FRAGMENT linear

(xi) SEQUENCE DESCRIPTION SEQ ID NO 34 GTAACCGTTG GCCCGGATAG CTCAGTCGGT AGAGCATCAG ACTTTTAATC TGAGGGTCCC 60

GCGTTCAAGT CCCTGTTCGG GCGCCACTGC TAGAGATTTT TTT 103

(2) INFORMATION ON SEQ ID NO 35

0) SEQUENCE LABEL

(A) LONG 103 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(H) MOLECULE TYPE Genome DNA (in) HYPOTHETICAL YES

(iv) ANTISENSE YES

(v) TYPE OF FRAGMENT linear

(xi) SEQUENCE DESCRIPTION SEQ ID NO 35 GTAACCGTTG GCCCGGATAG CTCAGTCGGT AGAGCATCAG ACTTTTAATC TGAGGGTCCA 60

AGGTTCAAGT CCCTGTTCGG GCGCCACTGC TAGAGATTTT TTT 103

(2) INFORMATION ON SEQ ID NO 36

0) SEQUENCE LABEL

(A) LONG 1 14 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(n) TYPE OF MOLECULE Genomic DNA (iii) HYPOTHETICAL: YES

(iv) ANTISENSE: YES

(v) TYPE OF FRAGMENT: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GTAACCGTTG GCCCGGATAG CTCAGTCGGT AGAGCATCAG ACTTTTAATC TGAGGGTCCC 60

GCGTTCAAGT CCCTGTTCGG GCGCCACTGC TAGAGACTGC TAGAGATTTT TTTT 114

(2) INFORMATION ON SEQ ID NO: 37:

(i) SEQUENCE LABEL:

(A) LENGTH: 1 14 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: YES

(iv) ANTISENSE: YES

(v) TYPE OF FRAGMENT: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GTAACCGTTG GCCCGGATAG CTCAGTCGGT AGAGCATCAG ACTTTTAATC TGAGGGTCCA 60

AGGTTCAAGT CCCTGTTCGG GCGCCACTGC TAGAGACTGC TAGAGATTTT TTTT 1 14

Claims

claims

1 . Nucleic acid construct, characterized in that the nucleic acid derived from a naturally occurring tRNA is extended at the 3 'end and the extended 3 ^' end is not or only partially complementary to the upstream nucleotides of a "primer binding site" (PBS Region).

2. Nucleic acid construct according to claim 1. characterized in that the 3 'end is extended by at least approximately 1 to 500, preferably by at least approximately 3-20, in particular by at least approximately 4-18, especially by at least approximately 14 to 18 nucleotides.

3. Nucleic acid construct according to one of claims 1-2, characterized in that the only partially complementary extended 3 ^' end at the 3' end of the extension is not complementary to a PBS-adjacent region or viral partial sequence.

4. Nucleic acid construct according to one of claims 1 -3, characterized in that the nucleic acid derived from a naturally occurring tRNA is additionally mutated.

5. Nucleic acid construct according to claim 4, characterized in that the mutation lies in the acceptor strain, D strain, anticodone strain and / or T strain of the nucleic acid derived from a naturally occurring tRNA, preferably in the T strain and / or D strain , especially in the T strain.

6. Nucleic acid construct according to claim 4 or 5, characterized in that the mutation is in the range of nucleotides 22-25 and / or 49-52, preferably in the range of nucleotides 49-52.

7. Nucleic acid construct according to any one of claims 4-6, characterized in that the mutation is a substitution, insertion or deletion.

8. Nucleic acid construct according to claim 7, characterized in that the substitution is selected from A23-> T. C25-> G, A50-> C,

G51 -> A or G52-> C, especially from A50-> C, G51 -> A or G52-> C, especially from A50-> C and G52-> C or G51 -> A.

9. Nucleic acid construct according to one of claims 1-8, characterized in that the naturally occurring tRNA is a virus-specific host tRNA, preferably tRNA ^{> s} ₃ .

10. Nucleic acid construct according to one of claims 1-9. characterized in that the construct is additionally derivatized.

1 1. Nucleic acid construct according to claim 10, characterized in that the nucleic acid construct is a phosphorothioate or a PNA derivative.

12. A method for producing a nucleic acid construct according to any one of claims 1 -1 1, characterized in that

(a) the nucleic acid construct is genetically, chemically or chemically synthesized and then optionally

(b) derivatized, (c) isolated and / or (d) purified.

13. Use of the nucleic acid constructs according to one of claims 1 -1 1 for the inhibition of reverse transcriptase.

14. Medicament containing a nucleic acid construct according to one of claims 1 -1 1 and, if appropriate, suitable additives and auxiliaries.

1 5. Use of a nucleic acid construct according to any one of claims 1 - 1 1 for the manufacture of a medicament for the therapy or prevention of a retroviral disease.

16. Use of a nucleic acid construct according to claim 1 5, characterized in that the drug for somatic

Gene therapy for retroviral diseases or for preventive protection against retroviral infections is used.

1 7. Use of a nucleic acid construct according to claim 15 or 16, characterized in that bone marrow cells of the patient are transformed in vitro with said nucleic acid construct, if appropriate, in a suitable expression vector.