WO1993006216A1 - Fusion proteins targeted to lysosomes, for the treatment of aids - Google Patents

Abstract

Fusion genes are constructed between a soluble CD4 (sCD4) gene, or portions thereof, and the genes of a lysosome targeting domain. Upon the biosynthesis of the fusion proteins in HIV infected cells, the sCD4 moiety binds newly synthesized gp160 and the lysosomal targeting moieties transport the entire complex to lysosomes. The genes diverts HIV coat glycoprotein to lysosomes for degradation, thus preventing the assembly of new virions and the propagation of HIV. The same process can be used for the treatment of other retroviruses.

Description

FUSION PROTEINS TARGETED TO LYSOSOMES, FOR THE TREATMENT OF AIDS

Background of the Invention

In 1981, acquired immune deficiency syndrome (AIDS) was identified as a disease that severely compromises the human immune system, and that almost without exception leads to death. In 1983, the etiological cause of AIDS was determined to be the human immunodeficiency virus (HIV). i December, 1990, the World Health Organization estimated that between 8 and 10 million people worldwide were infected with HTV, and of that number, between 1 ,000,000 and 1,400,000 were in the U.S.

There are at least two types of HIV, Type I and Type π. Both preferentially infect T4 helper T lymphocytes and macrophages by interacting with the molecule CD4 on the surface of the target cell. All viruses infect cells by binding to the cell of an envelope protein. In the case of HTV, the envelope protein is gpl20; the cell surface protein is an antigen called CD4. The viral membrane then apparently fuses with the cell membrane and the viral genes are injected into the cell, where they are replicated and new virions assembled using the host replicative processes. In some cases, the viral DNA most be integrated into the host genome, where it can remain latent for many years.

This replicative process has made it extremely difficult to treat, or as importantly, to cure HTV infection. Most attempts to vaccinate people against the disease have been unsuccessful; but at best would only limit infection. Most drugs have been targeted to replication of the viral nucleic acid.

In 1985, it was reported that the synthetic nucleoside 3'-azido-3'- deoxythymidine (AZT) inhibits the replication of human immunodeficiency virus type 1. Since then, a number of other synthetic nucleosides, including 2',3'-dideoxyinosine (DDI), 2',3'-dideoxycytidine (DDC), 3'-fluoro-3'-deoxythymidine (FLT), 2',3'-dideoxy,2\3'- didehydrothymidine (D4T), and 3'-azido-2',3'-dideoxyuridine (AZDU), have been demonstrated to be effective against HIV, although none are able to cure the disease nor do more than prolong the life expectancy of the infected individuals.

The only drug commercially available for the treatment of AIDS, 3^*-Azido-3'-deoxythymidine is a potent inhibitor of HIV reverse transcriptase. However, the benefits of AZT must be weighed against the severe adverse reactions of bone marrow suppression, nausea, myalgia, insomnia, severe headaches, anemia, peripheral neuropathy, and seizures. These adverse side effects often occur immediately after treatment begins, even though a minimum of six weeks of therapy is necessary to realize AZT's benefits. DDI, which has recently been approved.by the FDA for the clinical testing for the treatment of AIDS, is also associated with a number of side effects, including sporadic pancreatis and peripheral neuropathy. It is therefore apparent that there remains an important need to develop alternative therapies for treatment of HTV infections.

Gene therapy to achieve "intracellular immunization", as described by Baltimore, D. Nature 335: 395-396 (1988), against AIDS and other viral infections, especially of infections with retroviruses, offers definitive advantages because its successful application can potentially provide the patients with an intrinsic means to control the disease. To develop gene therapies for AIDS, it is equally important to generate new genes which can be used in AIDS therapy and to have the technology for gene transfer into the patients. Rapid advances in the technology for efficient gene transfer in vitro and in vivo have been made recently, for example, as reported by Friedmann, T. Science 244: 1275-1281 (1989). The developments of gene transfer vehicles, such as viral vectors, has led to clinical experimentation of gene therapy.

It is expected that the advances in gene therapy technology will continue. However, there are very few genes which have been demonstrated to be effective against HTV and potentially useful for gene therapy against AIDS. HTV glycoprotein gpl60 (precursor of gpl20 and gp41) is synthesized on polysomes and transported through the secretory pathway [endoplasmic reticulum (ER), Golgi, and secretory vesicles] to the cell surface where the assembly of new virions takes place. The strong binding of viral gpl20 to cell surface receptor CD4 is the primary route of HTV invasion of human cells (Klatzman, D., et al., Science 225: 59-63 (1984); Sattentau, Q.J., et al., Science 234: 1120-1123 (1986); Deen, K.C., et al., Nature 331: 82-84 (1988); Traunecher, A., et al., Nature 331: 84-86 (1988); Dalgleish, A.G., et al., Nature 312: 763-766 (1984); Maddon, P.J., et al., £eU 47: 333-348 (1986); Ho, D.D., et al., J. Clin. Invest. 77: 1712-1715 (1986); Gartner, S., et al., Science 233: 215-219 (1986)). The newly synthesized gρl60 in HIV-infected T lymphocyte cells can bind the newly synthesized CD4 molecules in the ER (Hoxie, J.A., et al., Science 234: 1123-1127 (1986); and Kawamura, L, et al., J. Viol. 63: 3748-3754 (1989)).

Buonocore and Rose constructed a modified sCD4 with an addition to the C-terminus of a 6-residue sequence (Sequence ID No. 5), SEKDEL, which is the signal for ER retention (Munro, S., and Pelham, H.R.B. Cell 48: 899-907 (1987)). The modified CD4, sCD4-KDEL, stayed in the ER and prevented the newly synthesized gpl60 from reaching the cell surface (Buonocore, L., and Rose, J.K. Nature 345: 625-628 (1990)). The ER residence of sCD4-KDEL has an clear limitation as a therapeutic agent, however. Proteins which reside in the ER, such as BiP, are transferred along the newly synthesized secretory proteins to the salvage compartment where the ER residence proteins are sorted and returned to ER (Pelham, H.R.B. Ann. Rev. Cell Biol. 5: 1- 23 (1989)). For sCD4-KDEL to be effective as an anti-HTV agent, it must be synthesized continuously and at a level higher than that for gpl60. This means that the continuing synthesis of sCD4-KDEL will ultimately exceed the capacity of sorting mechanism in the salvage compartment. After that point, the newly synthesized sCD4-KDEL and gpl60 will be lost from the ER, rendering sCD4-KDEL ineffective against HIV. It may also cause the loss of native ER residence proteins. Moreover, sCD4-KDE was not shown to resist HTV infection or propagation.

It is therefore an object of the present invention to provide a gene therapy for use in treating or preventing AIDS and other retroviral diseases.

Abstract of the Invention

This invention consists of the design and demonstration of fusion genes which can be used in the gene therapy for treating acquired immunodeficiency syndrome (AIDS) and other retroviruses. The principle of the therapeutic function is that upon transfer of these genes into human cells, the genes direct the synthesis of fusion proteins which interfere with the normal function of human immunodeficient virus (HIV), the causative agent of AIDS. The therapeutic genes are fusions between the genes encoding soluble CD4 (sCD4) (or other protein required for binding of virus to the target cell) and a lysosome targeting protein domain. Results have shown that when the fusion genes are expressed, the sCD4 moiety binds HTV glycoprotein gpl60 in the endoplasmic reticulum while the lysosome targeting moiety transports the entire complex to the lysosomes for degradation. Thus the therapeutic genes prevent gploO from reaching the cell surface, stopping the assembly of new virions and the propagation of HIV. The lysosome targeting domains successfully used in the fusion genes are human procathepsin D (PCaD) and parts of human lysosomal membrane proteins lamp-1, lamp-2, and acid phosphatase. The experimental evidences that established the predicted function of the new genes include the following: (a) the transfection of gpl60 gene into HeLa cells resulted in finding gpl60 protein on the cell surface. The transfection of one of the therapeutic genes with gpl60 genes into HeLa cells stopped gpl60 to reach the cell surface; (b) in the presence of one of the therapeutic genes in HeLa cells, gpl60 protein is degraded more rapidly than in the absence of the therapeutic gene; (c) the newly synthesized therapeutic proteins in HeLa cells are cleaved and digested in a manner characteristic of lysosomal activity; (d) transfecting one of the therapeutic genes into cultured T-lymphocyte cell line CEM inhibited the propagation of HTV.

Brief Description of the Drawings

Figure 1 is a schematic presentation of the overall strategy of the synthesis and secretion of proteins in the endoplasmic reticulum (ER) and Golgi. In ER, gpl60 of HTV is synthesized and glycosylated, and a fusion protein of soluble CD4-procathepsin D (sCD4-PCaD) is expressed from a cloned gene. The pro-cathepsin moiety of the sCD4-PCaD is N- glycosylated and mannose phosphorylated since it is a lysosomal enzyme. Then the gpl60 binds to the sCD4 moiety of sCD4-PCaD. The complex is transported through the cis-Golgi to trans-Golgi. In trans-Golgi network, the mannose-6-phosphate (Man-P0₄) receptors bind mannose-6- phosphate of the PCaD moiety and target the whole complex, including gpl60, to the lysosomes. In the lysosomes, procathepsin D activates to cathepsin D, and gploO and sCD4 moieties are proteolytically degraded. This strategy is designed to prevent gploO from entering the secretory pathway to reach the cell surface, thus preventing the assembly of HTV. Figure 2 is a schematic of the construction of sCD4-PCaD. The PCR primers used are: P-l, 5'-GAATTCAAGCCCAGAGCCCTGCC-3' (Sequence ID No. 6)

P-2, 5'-TCTAGAGGCCATTGGCTGCACCG-3' (Sequence ID

No. 7)

P-3, 5'-TCTAGACTCGTCAGGATCCCGCTG-3' (Sequence ID

No. 8)

P-4, 5'-GTCGACCTAGAGGCGGGCAGCC-3' (Sequence ID No. 9)

Figure 3 is a schematic of the construction of sCD4-HAP. The PCR primers used are:

P-l = 5'-TCTAGACAGCTGGCAAGCGGTCCTG-3' (Sequence ID No. 10)

P-2 = 5'-GTCGACTCAGGCGTGGTCCTCCCC-3' (Sequence ID No. 11) Figure 4 is a schematic of the construction of sCD4-Ll. The

PCR primers used are:

P-l = 5'-TCTAGACTGCTGGACGAGAACAGCAC-3' (Sequence^' ID No. 12)

P-2 = 5'-GTCGACACCAGGCTAGATAGTCTGGTAG-3' (Sequence ID No. 13)

Figure 5 is a. schematic of the construction of sCD4-L2. The

PCR primers used are:

P-l = 5'-TCTAGAAGTGCAGATGACGACAACTTC-3' (Sequence ID No. 14)

P-2 = 5'-GTCGACCTAAAATTGCTCATATCCAGCATG-3'

(Sequence ID No. 15)

Figure 6 is a graph of reverse rranscriptase (millions dpm/ml) versus time for blank vector pRc/RSV (1, -X-); no DNA (2, squares); pRc/RSV - sCD4-PCaD (3, inverted triangle); pRc/RSV - sCD4-HAP (4, -#-); pRc/RSV - sCD4-L2 (5, triangle); and pRc/RSV - sCD4-L2 (6, - diamond-). Figures 7A and B are graphs of β-hexosaminidase and density (g/ml) versus gradient fraction for Percoll gradients of lysosomes, showing the distribution of gpl60 in fractions from Percoll density gradient centrifugation in the presence and absence of sCD4-PCaD gene expression. Figure 7A shows the jS-hexosaminidase activity (solid line), density (broken line), and the autoradiography of the gel electrophoresis from cells transfected with gpl60 alone. Figure 7B are the same data from cells cotransfected with gpl60 and sCD4-PCaD genes.

Detailed Description of the Invention A method for treating retroviral infections, especially human immunodeficiency virus (HTV), wherein a fusion protein is created that binds to the viral envelope protein as it is formed and transports the envelope protein to lysosomes where it is degraded. The result is that the ability of the virus to replicate within the cell producing the fusion protein is limited. The fusion protein consists of two components: the protein which binds to the viral envelope protein and a protein (or domain of the protein) which targets the fusion protein to a lysosome. In the preferred embodiment for treating HTV, the first protein is soluble CD4, which binds HTV glycoprotein gpl60, and the second protein is procathepsin D (PCaD), parts of human lysosomal membrane proteins lamp-1, lamp-2, or acid phosphatase. In the most preferred embodiment, the fusion protein is sCD4-Ll or sCD4-L2.

The teachings of the following references cited herein are specifically incorporated herein as exemplifying methods or reagents useful in constructing and using the fusion proteins for treatment of viral disorders.

The sCD4 fusion proteins which bind to gploO and sort as a complex to lysosomes for degradation have the advantage over the ER retention shown by the modified sCD4 with an addition to the C-terminus of a 6-residue sequence (Sequence ID No. 5), SEKDEL, which is the signal for ER retention, of Buonocore and Rose, because of the continuing removal of bound gpl60 from the ER/Golgi system. sCD4 in the fusion gene can be substituted by its parts, domain Dl or combined domains D1-D2. sCD4, the extracellular segment of CD4, consists of four tandem immunoglobulin-like domains. The N- teπninal domain of sCD4, Dl, by itself binds gpl20 with high affinity, as reported by Arthos, J., et al. Cell 57, 469-481 (1989). Active recombinant Dl and D1-D2 domains have been obtained by (Arthos, et al.; Chao, B.H., et al., J. Biol. Chem. 264, 5812-5816 (1989);

Traunecker, A., et al., Nature 331, 84-86 (1988); and Berger, E., et al. Proc. Natl. Acad. Sci. USA. 85: 2357-2361 (1988)), indicating that these domains are capable of independent folding.

Two types of lysosomal targeting components are particularly suited for the fusion of sCD4. First is a lysosomal proenzyme which contains a structural marker for lysosomal targeting. Procathepsin D (PCaD) was chosen as the lysosome targeting domain of the prototype therapeutic gene for the reason that much is known of its structure and function relationships (Tang, J. and Wong, R.N.S. J. Cell. Biochem. 33: 53-63 (1987); Takahashi, T., et al., J. Biol. Chem.. 258: 2819-2830 (1983); Shewale, J.G., and Tang, J. Proc. Natl. Acad. Sci. USA. 80: 3703-3707 (1984); Yonezawa, S., et al., J. Biol. Chem. 263: 16504- 16511 (1988); Faust, P.L., et al., Proc. Natl. Acad. Sci. USA. 82: 4910- 4914 (1985)), its sorting mechanism via mannose-6-phosphate receptors in the trans-Golgi network (Kornfeld, S. and Mellman, I. Ann. Rev. Cell Biol. 5: 483-525 (1989)), and the spontaneous activation of its precursor, procathepsin D, in the lysosomes (Hasilick, A., et al., Eur. J. Biochem. 125: 37-321 (1982)).

The second type of lysosomal targeting domain for sCD4 fusion is taken from part of the lysosomal membrane proteins. Three human lysosomal membrane proteins, lamp-1 (LI), lamp-2 (L2), and lysosomal acid phosphatase (HAP) (Fukuda, M., et al., J. Biol. Chem. 263: 18920- 18928 (1988); Pohlmann, R. et al. EMBO J. 7: 2343-2350 (1988); and Waheed, A. et al. ibid. 7: 2351-2358 (1988)); each contains in its C- terminus a "lysosomal targeting signal" (LTS) region which consists of a short membrane-anchoring sequence and a short cytosolic domain (Kornfeld, S. and Mellman, I. Ann. Rev. Cell Biol. 5: 483-525 (1989); Peters, C, et al., EMBO J. 9: 3497-3506 (1990); William, M.A. and Fukuda, M. J. Cell Biol. Il l : 955-966 (1990)). These three sCD4-LTS fusion genes, sCD4-HAP, sCD4-Ll, and sCD4-L2, form the second group of therapeutic genes. The basic function of the sCD4-LTS therapeutic genes is the same as that in sCD4-PCaD, even though the targeting mechanisms are different between these two groups.

The PCaD moiety can be substituted for by other soluble lysosomal enzymes, such as other lysosomal soluble proteins containing mannose-6-phosphate markers. Some examples of human lysosomal enzymes with known cDNA structures and their cDNA sizes are α-N- Acetylgakctosaminidase, 1.3 kbp (Wang, A.M., et al., J. Biol. Chem. 265: 21859-21866 (1990)); Glycosylasparaginase, 1.1 kbp (Fisher, K.J., et al., FEBS Lett. 276: 440-444 (1990)); Glucocerebrosidase, 1.8 kbp

(Tsuji, S., et al., J. Biol. Chem. 261: 50-53 (1986)); Procathepsin L, 1.1 kbp (Gal, S. and Gottesman, M.M. Biochem. J. 253: 303-306 (1988)); Procathepsin B, 1.1 kbp (Chan, S.J., et al., Proc. Nad. Acad. Sci. USA 83: 7721-7725 (1986)); and Procathepsin E, 1.2 kbp (Azuma, T., et al., J. Biol. Chem. 264: 16748-16753 (1986)). The lysosomal targeting domains (the transmembrane domains and cytosolic domains) of other lysosomal membrane proteins (human or other species origins) can also be substituted for human HAP, LI and L2 in the therapeutic fusion genes. It is believed that it will be possible to use more than one lysosome targeting domain in a therapeutic gene. Multiple targeting domains can increase the efficiency and capacity of the gpl60 transporting to lysosomes. Examples of multidomain therapeutic genes are sCD4-PCaD-HAP, sCD4-PCaD-PCaD, and other combinations. The linker peptide between the sCD4 and the lysosome targeting domain can also be altered in arnino acid sequence and in length to achieve different efficiencies and capacity of gpl60 transporting to lysosomes.

The gene encoding the fusion protein is constructed using standard genetic engineering techniques, as described in detail below for treatment of HTV. The fusion gene is then introduced into cells in vitro using methods such as calcium phosphate coprecipitation, lipofection (liposomes), cell fusion, electroporation, or a vector such as a vaccinia virus. For example, CEM-SS cells were grown to exponential phase. Plasmids containing 10 μg each of the sCD4-LTD genes were separately transformed into 2 x 10⁷ cells by electroporation using the method of Aldovini, A. and M.B. Feinberg, Techniques in HTV Research eds. Aldovini, A. and B.D. Walker, pp. 147-176 (Stockton Press, NY 1990).

For in vivo applications, the preferred method is by transfection using a vector system such as the vaccinia virus. The vector is not limited however, for efficient expression, mammalian or viral promoters and other regulatory elements must be present. A recent account of the progress in gene therapy of humans is described in Friedmann, T., Science 244: 1275-1281 (1989). Some vector systems for introduction of therapeutic genes into AIDS patients have been described by Chimada, T., et al., J. Clin. Invest. 88:1043-1047.

The following examples demonstrate the effectiveness of the method for treating HTV infection. However, the same strategy could be used with other retroviral infections, such as hepatitis B and HTLV-1 caused leukemia. these diseases, the cell surface protein receptors which bind virus glycoprotein can be used in fusion with lysosome targeting protein.

The present invention will be further understood by reference to the following non-limiting examples of the construction and cloning of therapeutic fusion genes, and their efficacy in degrading HTV envelope protein as it is formed.

Example 1: Construction and Cloning of the Therapeutic Genes.

Four sCD4-fusion genes, sCD4-PCaD, sCD4-HAP, sCD4-Ll , and sCD4-12, were constructed as shown in the following sequences.

Schematics of their constructions are shown in Figures 2-5, respectively. The gene encoding human procathepsin D cDNA was reported by Faust, et al., (1985). The gene encoding human lamp-1 and lamp-2 was reported by Fukuda, et al., (198). The gene encoding human HAP was reported by Pohlmann, et al., (1988).

The nucleotide sequence encoding sCD4-PCaD is shown below in Sequence TD No. 1. Underlined letters are engineered restriction sites.

GAATTCAAGC CCAGAGCCCT GCCATTTCTG TGGGCTCAGG TCCCTACTGC TCAGCCCCTT CCTCCCTCGG CAAGGCCACA ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG GGACCAAGGA AACTTCCCCC TGATCATCAA GAATCTTAAG ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC AGAAGGAGGA GGTGCAATTG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC AATGTAGGAG TCCAAGGGGT AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC TGGAGCTCCA GGATAGTGGC ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG AGTTCAAAAT AGACATCGTG GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA AAGAGGGGGA ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT TTGACCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA TGGGCAAGAA GCTCCCGCTC CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT CTGGAAACCT CACCCTGGCC CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC TGGTGGTGAT GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC GGGAGAAGGC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG ACTCGGGACA GGTCCTGCTG GAATCCAACA TCAAGGTTCT GCCCACATGG TCCACCCCGG TGCAGCCAAT GGCCTCTAGA CTCGTCAGGA TCCCGCTGCA CAAGTTCACG TCCATCCGCC GGACCATGTC GGAGGTTGGG GGCTCTGTGG AGGACCTGAT TGCCAAAGGC CCCGTCTCAA AGTACTCCCA GGCGGTGCCA GCCGTGACCG AGGGGCCCAT TCCCGAGGTG CTCAAGAACT ACATGGACGC CCAGTACTAC GGGGAGATTG GCATCGGGAC GCCCCCCCAG TGCTTCACAG TCGTCTTCGA CACGGGCTCC TCCAACCTGT GGGTCCCCTC CATCCACTGC AAACTGCTGG ACATCGCTTG CTGGATCCAC CACAAGTACA ACAGCGACAA GTCCAGCACC TACGTGAAGA ATGGTACCTC GTTTGACATC CACTATGGCT CGGGCAGCCT CTCCGGGTAC CTGAGCCAGG ACACTGTGTC GGTGCCCTGC CAGTCAGCGT CGTCAGCCTC TGCCCTGGGC GGTGTCAAAG TGGAGAGGCA GGTCTTTGGG GAGGCCACCA AGCAGCCAGG CATCACCTTC ATCGCAGCCA AGTTCGATGG CATCCTGGGC ATGGCCTACC CCCGCATCTC CGTCAACAAC GTGCTGCCCG TCTTCGACAA CCTGATGCAG CAGAAGCTGG TGGACCAGAA CATCTTCTCC TTCTACCTGA GCAGGGACCC AGATGCGCAG CCTGGGGGTG AGCTGATGCT GGGTGGCACA GACTCCAAGT ATTACAAGGG TTCTCTGTCC TACCTGAATG TCACCCGCAA GGCCTACTGG CAGGTCCACC TGGACACAGG CACTTCCCTC ATGGTGGGCC CGGTGGATGA GGTGCGCGAG CTGCAGAAGG CCATCGGGGC CGTGCCGCTG ATTCAGGGCG AGTACATGAT CCCCTGTGAG AAGGTGTCCA CCCTGCCCGC GATCACACTG AAGCTGGGAG GCAAAGGCTA CAAGCTGTCC CCAGAGGACT ACACGCTCAA GGTGTCGCAG GCCGGGAAGA CCCTCTGCCT GAGCGGCTTC ATGGGCATGG ACATCCCGCC ACCCAGCGGG CCACTCTGGA TCCTGGGCGA CGTCTTCATC GGCCGCTACT ACACTGTGTT TGACCGTGAC AACAACAGGG TGGGCTTCGC CGAGGCTGCC CGCCTCTAGC AGCTG

The nucleotide sequence encoding sCD4-HAP is shown below in Sequence ID No. 2. Underlined letters are engineered restriction sites.

GAATTCAAGC CCAGAGCCCT GCCATTTCTG TGGGCTCAGG TCCCTACTGC TCAGCCCCTT CCTCCCTCGG CAAGGCCACA ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG GGACCAAGGA AACTTCCCCC TGATCATCAA GAATCTTAAG ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC AGAAGGAGGA GGTGCAATTG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC AATGTAGGAG TCCAAGGGGT AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC TGGAGCTCCA GGATAGTGGC ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG AGTTCAAAAT AGACATCGTG GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA AAGAGGGGGA ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT TTGACCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA TGGGCAAGAA GCTCCCGCTC CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT CTGGAAACCT CACCCTGGCC CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC TGGTGGTGAT GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC GGGAGAAGGC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG ACTCGGGACA GGTCCTGCTG GAATCCAACA TCAAGGTTCT GCCCACATGG TCCACCCCGG TGCAGCCAAT GGCCTCTAGA CAGCTGGCAA GCGGTCCTGC AGACACAGAG GTGATTGTGG CCTTGGCTGT ATGTGGCTCC ATCCTCTTCC TCCTCATAGT GCTGCTCCTC ACCGTCCTCT TCCGGATGCA GGCCCAGCCT CCTGGCTACC GCCACGTCGC AGATGGGGAG GACCACGCCT GAGTCGAC

The nucleotide sequence encoding sCD4-Ll is shown below in Sequence TD No. 3. Underlined letters are engineered restriction sites.

GAATTCAAGC CCAGAGCCCT GCCATTTCTG TGGGCTCAGG TCCCTACTGC TCAGCCCCTT CCTCCCTCGG CAAGGCCACA ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG GGACCAAGGA AACTTCCCCC TGATCATCAA GAATCTTAAG ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC AGAAGGAGGA GGTGCAATTG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC AATGTAGGAG TCCAAGGGGT AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC TGGAGCTCCA GGATAGTGGC ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG AGTTCAAAAT AGACATCGTG GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA AAGAGGGGGA ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT TTGACCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA TGGGCAAGAA GCTCCCGCTC CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT CTGGAAACCT CACCCTGGCC CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC TGGTGGTGAT GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC GGGAGAAGGC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG ACTCGGGACA GGTCCTGCTG GAATCCAACA TCAAGGTTCT GCCCACATGG TCCACCCCGG TGCAGCCAAT GGCCTCTAGA CTGCTGGACG AGAACAGCAC GCTGATCCCC ATCGCTGTGG GTGGTGCCCT GGCGGGGCTG GTCCTCATCG TCCTCATCGC CTACCTCGTC GGCAGGAAGA GGAGTCACGC AGGCTACCAG ACTATCTAGC CTGGTGTCGA C

The nucleotide sequence encoding sCD4-L2 is shown below in Sequence ID No. 4. Underlined letters are engineered restriction sites.

GAATTCAAGC CCAGAGCCCT GCCATTTCTG TGGGCTCAGG TCCCTACTGC TCAGCCCCTT CCTCCCTCGG CAAGGCCACA ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG GGACCAAGGA AACTTCCCCC TGATCATCAA GAATCTTAAG ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC AGAAGGAGGA GGTGCAATTG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC AATGTAGGAG TCCAAGGGGT AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC TGGAGCTCCA GGATAGTGGC ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG AGTTCAAAAT AGACATCGTG GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA AAGAGGGGGA ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT TTGACCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA TGGGCAAGAA GCTCCCGCTC CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT CTGGAAACCT CACCCTGGCC CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC TGGTGGTGAT GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC GGGAGAAGGC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG ACTCGGGACA GGTCCTGCTG GAATCCAACA TCAAGGTTCT GCCCACATGG TCCACCCCGG TGCAGCCAAT GGCCTCTAGA AGTGCAGATG ACGACAACTT CCTTGTGCCC ATAGCGGTGG GAGCTGCCTT GGCAGGAGTA CTTATTCTAG TGTTGCTGGC TTATTTTATT GGTCTCAAGC ACCATCATGC TGGATATGAG CAATTTTAGG TCGAC

Example 2: Demonstration of efficacy of the Therapeutic Genes:

I. Hela-CD4+ Cells Expressing GplόO Formed Syncytia which was Reversed by Coexpressing of Therapeutic Genes. HeLa-CD4+ cell are HeLa cells which express CD4 on the cell surface. When the gpl60 gene is transfected into HeLa-CD4+ cells, the cells have both CD4 and gpl60 on the cell surface. Because CD4 binds gpl60 tightly, the cells aggregate and fuse together to form giant cell masses called synthetium. When gploO and a therapeutic gene are both transfected into the cells, even though die gpl60 is synthesized, it does not appear on the cell surface because the protein made from the therapeutic gene transports the gpl60 to die lysosomes instead of the cell surface, so no syncytium is formed.

Syncytium formation was monitored for HeLa-CD4+ cells (clone HT4-6C) transfected with gpl60 or cotransfected with gpl60 and ouier genes, using a vaccinia virus expression system. The following results were obtained.

Untransfected cells No syncytium

Cells transfected with procathepsin D (control) No syncytium

Cells transfected with gpl60 gene Syncytia

Cells cotransfected with gpl60 and sCD4-PCaD gene in die wrong cloning direction Syncytia

Cells cotransfected with gpl60 and blank pET-3a vector Syncytia

Cells cotransfected with gpl60 and therapeutic gene sCD4-PCaD (right direction) No syncytium Cells cotransfected with gpl60 and therapeutic gene sCD4-HAP No syncytium

Cells cotransfected with gpl60 and sCD4 Syncytia

Cells cotransfected with gpl60 and sCD4-Ll No syncytium

Cells cotransfected with gpl60 and sCD4-L2 No syncytium

These results were completely reproducible in tiiree experiments using HeLa-CD4+ clone 6C and in one experiment with clone 1022.

These results indicate that neitiier sCD4 nor the control vectors could reverse the syncytium formation. Only the therapeutic genes sCD4-

PCaD and sCD4-HAP can reverse die syncytium formation by gpl60.

The expression of the lysosome targeting domain wim me sCD4 is necessary to prevent die syncytium.

II. Gpl60 and the Therapeutic Gtene Products (sCD4-PCaD and sCD4-HAP proteins) and gpl60 are Degraded in the Lysosomes.

The following reagents used in these studies were obtained through die AIDS Research and Reference Program, AIDS Program,

NIATD, NTH: plasmid pHIenv3-l (reagent #289, from Dr. Joseph

Sodroski), plasmid pt4B (#157, from Dr. Richard Axel), antiserum to

HIV-1 gpl20 (#288, from Dr. Michael Phelan), antiserum to CD4 (#314, from Dr. Michael Phelan), vector vTF7-3 (#356, from Drs. Tom Fuerst and Bernard Moss), HeLa cells (#153, from Dr. Richard Axel), HeLa-

CD4+ (clone 6C) (#459, from Dr. Bruce Chesebro), HeLa-CD4+ (clone

1022) (#1109, from Dr. Bruce Chesebro), and CEM-SS cells (#776, from

Dr. Peter L. Nara). Rabbit anticathepsin D antiserum was produced as described by Huang, J.S. et al., J. Biol. Chem. 254, 11405-11417 (1979). HeLa cells were transfected witii gpl60 gene or cotransfected witii gploO and one of die tiierapeutic genes (sCD4-PCaD or sCD4-HAP), using a vaccinia expression system for transfection and expression. The proteins were metabolically labelled with radioactive methiorύne (pulse). At different times (chase), die cells were separated from die culture medium and homogenized. Immunoprecipitations were performed on cell homogenates and media with antibodies against gpl60, CD4, and PCaD, respectively. The precipitated proteins were separated on SDS-gel electrophoresis and ti eir patterns visualized by exposure to photographic films.

The pattern of die specific proteins indicate die fates of various gploO and die therapeutic proteins. The results from d e pulse-chase experiments indicate mat gploO is not degraded when e gene for gploO is transfected alone. When gploO and d e tiierapeutic genes are cotransfected, both proteins are rapidly degraded. The results are summarized below.

The pattern of protein bands immunoprecipitated widi gpl60 antibody after a 4 h chase when gpl60 was transfected alone, showed that both gploO and gpl20 bands were associated with die cells; but only gpl20 was in die medium due to shedding from cell surface.

Cotransfection of gploO and sCD4 did not significantly change from gpl60 alone. When gploO was cotransfected with either sCD4-PCaD gene (sCD4-P) or sCD4-HAP gene (sCD4-H), no gpl20 was observed either in die cells or in the media, indicating mat gpl60 did not enter into die patiiway to die cell surface which also processes gpl60 to gpl20 and gp41. A band was observed at near 96 kD of die cells witii sCD4- PCaD cotransfection. This band represents metabolically radiolabeled sCD4-PCaD which binds strongly to gploO and coprecipitates by antibody against gpl60. Similarly, a 50-kD band from d e cells cotransfected with sCD4-HAP gene represents die fusion protein sCD4- HAP. These results have been completely reproducible in several experiments.

The results demonstrate tiiat sCD4-fusion genes cause gploO to go to a different transporting pathway. Strong bindings between sCD4- fusion proteins and gpl60 takes place in die cells.

The patterns from immunoprecipitation of cell homogenates and media with antibodies against gpl60 obtained by SDS-gel electrophoresis also demonstrate tiiat gpl60 is degraded more rapidly in die presence of sCD4-PCaD gene. After a 4 h chase, die combined amount of gploO and gpl20 in die cells transfected with only gploO gene already far exceeds the gpl60 band from die cells cotransfected with gpl60 and sCD4-PCaD genes.

After an 18 h chase, die amount of combined gpl60 and gpl20 (gpl60 transfection) did not appreciably change from die 4 h result. However, die gplδO band is largely diminished in die cells cotransfected with die sCD4-PCaD fusion gene. (The intensity of this gpl60 band is only about 1/8 of tiiat of the combined gploO + gpl20.) These results indicate gpl60 in die cells synthesizing sCD4-PCaD is rapidly degraded as a result of die expression of die sCD4-PCaD gene in die cells. This conclusion is consistent with die explanation that gpl60 is transported by the fusion protein to lysosomes and degraded.

Cathepsin D antibody immunoprecipitates show similar results by SDS gel electrophoresis. At die end of a pulse or after a 2 h chase, a major band near 97 kD is clearly die fusion protein sCD4-PCaD. The molecular size of this band agrees with die fusion between sCD4

(apparent size: 46 kD) and procathepsin D (apparent size: 50 kD). With a 4 h chase, cathepsin D appeared mainly as a 35 kD band. This is close to die size of the human cathepsin D heavy chain, usually in the range of 30 to 35 kD. (The 0.9 kD light chain is usually too faint to be seen.) It is known that procathepsin is rapidly activated to cathepsin D and slowly processed to a 2-chain enzyme in die lysosomes, as reported by Hasilik, A. and Neufeld, E.F. J. Biol. Chem. 255: 4937-4945 (1980) and Erickson, A.H., et al., J. Biol. Chem. 256: 11224-11231 (1981). Thus, me time for the fusion protein to reach lysosomes is between 2 to 4 h. In summary, the sCD4-PCaD fusion protein is targeted to the lysosomes, activated, and correcdy processed.

The electrophoretic patterns of CD4 antibody immunoprecipitates at different chase times also show similar results. At the end of pulse labelling of 20 min (0 chase time), gpl60 transfection alone provides only faint background bands, as no CD4 synthesis is expected in HeLa cells. Cotransfection with sCD4-PCaD produces a major band near 97 kD. This is die same position for d e synthetic band detected with cathepsin D antibody. Even as early as at the end of die 20 min pulse period, some degradation bands of sCD4 could be seen, witii die 46 kD band appearing at die same position as die authentic sCD4; so this band must have come from die activation of die procathepsin D moiety of die fusion protein in the lysosomes which resulted in die separation of sCD4 from PCaD. The major band for die cotransfection of sCD4-HAP of 0 time chase is about 50 kD. Since die lysosome targeting domain from HAP is only 45 amino acid residues, this is d e expected size for die fusion protein sCD4-HAP.

After a 2 h chase, much of the labelled sCD4- domain in sCD4- PCaD had been degraded to 3 bands in die size range of 25 to 40 kD. The sCD4-HAP band had become a doublet due to die degradation of die targeting domain from the C-terminal of sCD4, which is known to occur for all die lysosomal membrane proteins. After 4 h and 16 h chase periods, diminishing amounts of material recognizable by CD4 antibody were present due to further lysosomal proteolysis.

In conclusion, some sCD4-PCaD fusion protein reaches lysosomes as early as 20 min. At d e end of 2 h, die degradation of sCD4-fusion proteins is extensive. At 16 h, the degradation is nearly complete. The degradation kinetics of sCD4- moiety is similar to that of gpl60 which was nearly completed after 18 h.

TJLT. T-lymphocyte CEM cells transfected with therapeutic genes can resist HTV propagation.

CEM(CD4+) cells are T-lymphoma cells which have CD4 on tiieir cell surface. These cells are susceptible for HIV infection. The cells are transfected with therapeutic genes which transiently expressing therapeutic proteins. The transfected cells, control cells transfected witii blank vectors, and untransfected cells are challenged with HTV. HTV propagation was determined by die analysis of reverse transcriptase activity.

As shown in Figure 6, reverse transcriptase (RT) activities at different time in control cells (electroporation witiiout DNA), in cells separately transfected witii blank vector, and vectors witii one of four therapeutic genes (sCD4-PCaD, sCD4-HAP, sCD4-Ll and sCD4-L2).

Transfections were done 40 h before die addition of HTV (day 0). Up to day 3, no difference could be seen in four cell groups. However, at day

7, die control cells and die cells transfected with blank vector botii showed a dramatic increase in RT activity. The agreements among these two controls are reasonably good. tiie cells transfected witii sCD4- fusion genes, however, die increases are only about 10-15% of the controls.

The transient expression of die fusion genes usually peaks around two to tiiree days and lasted for several days. However, die differences in RT activity became apparent at day 7. This means that from day 0 to day 3 die sCD4-fusion genes are depriving die HTV virions of gpl60

(gpl20); tiius, the deficiency of gploO on tiie virions was manifested as inhibition of propagation at day 7. These data support the view that the fusion genes work as intended in CEM cells challenged with HIV. The results lead to d e following conclusions: Therapeutic genes can reverse syncytium formations. Thus, die therapeutic genes prevent gpl60 from reaching cell surface. When the sCD4-fusion genes are expressed, gpl60 and sCD4 are degraded rapidly. All evidence shows die main site of degradation is die lysosomes. When sCD4-PCaD is expressed, procathepsin D moiety is activated and correctly processed, which is exclusively a lysosomal phenomenon. Thus, sCD4-PCaD protein enters lysosomes. In summary,, it can be concluded tiiat die therapeutic genes transport gpl60 to lysosomes. Therapeutic genes sCD4-PCaD, sCD4-HAP, sCD4-Ll, and sCD4-L2 inhibit HTV propagation in T-lymphocyte cell line CEM cells. Example 3: Expression of sCD4-PCaD gene causes gpl60 to be present in the lysosomes. To further demonstrate sCD4-fusion proteins are degraded in the lysosomes and tiiat gpl60 is transported to die lysosomes by each of d e 4 sCD4 fusion proteins, lysosomes were fractionated in Percoll density gradient centrifugation from the ³⁵S-metiιionine labelled cells which had been either transfected with gpl60 or cotransfected with gpl60 and sCD4-PCaD genes. HeLa cells were transfected witii gpl60 gene or cotransfected with gρl60 + sCD4-PCaD genes, labelled with ^3SS- methionine, and chased for 4 h as described in die pulse-chase experiments. Cells were scraped from plates, homogenized, and centrifuged to obtain die postnuclear supernate as described by Gieselmann, J. Cell Bio 97: 1-5 (1983). This supernate (1 ml) was applied as a top layer of a 15 % Percoll (Pharmacia) solution of 0.25 M sucrose in a Beckman polycarbonate centrifuge bottle (No. 355603) and centrifuged in a Beckman 80Ti rotor at 33,000 x g for 30 min. Fractions of 0.75 ml each, which were collected starting from the bottom, were, analyzed for /3-hexosaminidase activity (Geiger and Arnon, Metiiods in Enzvmologv 50, 547-555 (1978) and were subjected to immunoprecipitation using anti-gpl60 antiserum. The electrophoresis of precipitate and autoradiography are die same as described above. Figure 7A shows die β-hexosaminidase activity (solid line), density (broken line), and die autoradiography of die gel electrophoresis from cells transfected witii gploO alone. Figure 7B are die same data from cells cotransfected with gpl60 and sCD4-PCaD genes.

The fractions were analyzed for lysosomes by β-hexosaminidase activity and also immunoprecipitated with anti-gpl60 antiserum, electrophoresed, and visualized by autoradiography. Figures 7A and B shows that the activity of hexoaminidase accumulates in fractions 1-3, which represent dense lysosomes, and is more pronounced in fractions 10-13, which represent the light lysosomes and endosomes. The total β- hexosaminidase activates are similar in two transfections (Figures 7A and B), meaning that the number of lysosomes are about die same in diem. However, gplδO bands are present only in the cells cotransfected witii sCD4-PCaD gene, not in die cells transfected with gpl60 alone. These observations support die view tiiat die expression of sCD4-PCaD gene diverts gpl60 to lysosomes.

Example 4: Demonstration that the fusion gene can encode portions of the binding protein and be efficacious.

Dl and D1-D2 were tested as alternatives of the sCD4 domain in die sCD4-fusion genes. The results from syncytium formation and pulse- chase experiments indicated that sCD4 in die fusion gene can be substituted by its parts, domain Dl or combined domains D1-D2. The ability of Dl-HAP and D1-D2-HAP to reverse the syncytium formation caused by die transfection of gpl60 into HeLa-CD4+ cells. The experimental conditions are the same as already described for the other syncytium experiments. It was observed tiiat botii fusion genes, when cotransfected with gpl60 gene, prevented die syncytium formation. Pulse-chase studies using the fusion genes were also conducted in HeLa cells. Antiserum against gpl60 was used to immunoprecipitate. It was observed tiiat Dl-HAP and D1-D2-HAP coprecipitated witii gpl60.

These observations suggest tiiat Dl and D1-D2 fusion genes work as effectively as the sCD4 fusion genes.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Tang, J. N.

(ii) TITLE OF INVENTION: Pusion Protein Genes for Treatment of Aids

(iii) NUMBER OF SEQUENCES: 15

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Kilpatric & Cody

(B) STREET: 1100 Peachtree Street

(C) CITY: Atlanta

(D) STATE: Georgia

(E) COUNTRY: U.S.

(F) ZIP: 30309-4530

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

( i) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Pabst, Patrea L.

(B) REGISTRATION NUMBER: 31,284

(C) REFERENCE/DOCKET NUMBER: OMRF129

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 404-815-6508

(B) TELEFAX: 404-815-6555 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2465 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapien (F) TISSUE TYPE: Epithelial

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..6

(D) OTHER INFORMATION: /note= "Restriction site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 2460..2465

(D) OTHER INFORMATION: /note= "Restriction site"

(xi) SEQUENCE DESCRIPTION: SEQ ID NOtl: GAATTCAAGC CCAGAGCCCT GCCATTTCTG TGGGCTCAGG TCCCTACTGC TCAGCCCCTT 60 CCTCCCTCGG CAAGGCCACA ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC 120 TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG 180 GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA 240 AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT 300 CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG GGACCAAGGA AACTTCCCCC 360 TGATCATCAA GAATCTTAAG ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC 420 AGAAGGAGGA GGTGCAATTG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC 480 TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC 540 AATGTAGGAG TCCAAGGGGT AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC 600 TGGAGCTCCA GGATAGTGGC ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG 660 AGTTCAAAAT AGACATCGTG GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA 720 AAGAGGGGGA ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG 780

GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT 840

TTGACCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA 900

TGGGCAAGAA GCTCCCGCTC CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT 960

CTGGAAACCT CACCCTGGCC CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC 1020

TGGTGGTGAT GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA 1080

CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC 1140

GGGAGAAGGC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG 1200

ACTCGGGACA GGTCCTGCTG GAATCCAACA TCAAGGTTCT GCCCACATGG TCCACCCCGG 1260

TGCAGCCAAT GGCCTCTAGA CTCGTCAGGA TCCCGCTGCA CAAGTTCACG TCCATCCGCC 1320

GGACCATGTC GGAGGTTGGG GGCTCTGTGG AGGACCTGAT TGCCAAAGGC CCCGTCTCAA 1380

AGTACTCCCA GGCGGTGCCA GCCGTGACCG AGGGGCCCAT TCCCGAGGTG CTCAAGAACT 1440

ACATGGACGC CCAGTACTAC GGGGAGATTG GCATCGGGAC GCCCCCCCAG TGCTTCACAG 1500

TCGTCTTCGA CACGGGCTCC TCCAACCTGT GGGTCCCCTC CATCCACTGC AAACTGCTGG 1560

ACATCGCTTG CTGGATCCAC CACAAGTACA ACAGCGACAA GTCCAGCACC TACGTGAAGA 1620

ATGGTACCTC GTTTGACATC CACTATGGCT CGGGCAGCCT CTCCGGGTAC CTGAGCCAGG 1680

ACACTGTGTC GGTGCCCTGC CAGTCAGCGT CGTCAGCCTC TGCCCTGGGC GGTGTCAAAG 1740

TGGAGAGGCA GGTCTTTGGG GAGGCCACCA AGCAGCCAGG CATCACCTTC ATCGCAGCCA 1800

AGTTCGATGG CATCCTGGGC ATGGCCTACC CCCGCATCTC CGTCAACAAC GTGCTGCCCG 1860

TCTTCGACAA CCTGATGCAG CAGAAGCTGG TGGACCAGAA CATCTTCTCC TTCTACCTGA 1920 •

GCAGGGACCC AGATGCGCAG CCTGGGGGTG AGCTGATGCT GGGTGGCACA GACTCCAAGT 1980

ATTACAAGGG TTCTCTGTCC TACCTGAATG TCACCCGCAA GGCCTACTGG CAGGTCCACC 2040

TGGACCAGGT GGAGGTGGCC AGCGGGCTGA CCCTGTGCAA GGAGGGCTGT GAGGCCATTG 2100

TGGACACAGG CACTTCCCTC ATGGTGGGCC CGGTGGATGA GGTGCGCGAG CTGCAGAAGG 2160

CCATCGGGGC CGTGCCGCTG ATTCAGGGCG AGTACATGAT CCCCTGTGAG AAGGTGTCCA 2220

CCCTGCCCGC GATCACACTG AAGCTGGGAG GCAAAGGCTA CAAGCTGTCC CCAGAGGACT 2280

ACACGCTCAA GGTGTCGCAG GCCGGGAAGA CCCTCTGCCT GAGCGGCTTC ATGGGCATGG 2340 ACATCCCGCC ACCCAGCGGG CCACTCTGGA TCCTGGGCGA CGTCTTCATC GGCCGCTACT 2400 ACACTGTGTT TGACCGTGAC AACAACAGGG TGGGCTTCGC CGAGGCTGCC CGCCTCTAGC 2460 AGCTG 2465

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1448 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapien (F) TISSUE TYPE: Epithelial

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..6

(D) OTHER INFORMATION: /note= "Restriction site"

(ix) FEATURE:

(A) NAME/KEYt misc_feature

(B) LOCATION: 1275..1280

(D) OTHER INFORMATION: /note= "Restriction site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1444..1448

(D) OTHER INFORMATION: /note= "Restriction site"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GAATTCAAGC CCAGAGCCCT GCCATTTCTG TGGGCTCAGG TCCCTACTGC TCAGCCCCTT 60 CCTCCCTCGG CAAGGCCACA ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC 120 TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG 180 GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA 240 AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT 300 CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG GGACCAAGGA AACTTCCCCC 360 TGATCATCAA GAATCTTAAG ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC 420 AGAAGGAGGA GGTGCAATTG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC 480 TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC 540 AATGTAGGAG TCCAAGGGGT AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC 600 TGGAGCTCCA GGATAGTGGC ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG 660 AGTTCAAAAT AGACATCGTG GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA 720 AAGAGGGGGA ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG 780 GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT 840 TTGACCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA 900 TGGGCAAGAA GCTCCCGCTC CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT 960 CTGGAAACCT CACCCTGGCC CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC 1020 TGGTGGTGAT GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA 1080 CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC 1140 GGGAGAAGGC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG 1200 ACTCGGGACA GGTCCTGCTG GAATCCAACA TCAAGGTTCT GCCCACATGG TCCACCCCGG 1260 TGCAGCCAAT GGCCTCTAGA CAGCTGGCAA GCGGTCCTGC AGACACAGAG GTGATTGTGG 1320 CCTTGGCTGT ATGTGGCTCC ATCCTCTTCC TCCTCATAGT GCTGCTCCTC ACCGTCCTCT 1380 TCCGGATGCA GGCCCAGCCT CCTGGCTACC GCCACGTCGC AGATGGGGAG GACCACGCCT 1440 GAGTCGAC 1448

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1421 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapien (F) TISSUE TYPE: Epithelial

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..6

(D) OTHER INFORMATION: /note= "Restriction site"

(ix) FEATURE:

(A) NAME/KEY: miβc_feature

(B) LOCATION: 1275..1280

(D) OTHER INFORMATION: /note= "Restriction site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1416..1421

(D) OTHER INFORMATION: /note= "Restriction site"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAATTCAAGC CCAGAGCCCT GCCATTTCTG TGGGCTCAGG TCCCTACTGC TCAGCCCCTT 60 CCTCCCTCGG CAAGGCCACA ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC 120 TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG 180 GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA 240 AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT 300 CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG GGACCAAGGA AACTTCCCCC 360 TGATCATCAA GAATCTTAAG ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC 420 AGAAGGAGGA GGTGCAATTG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC 480 TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC 540 AATGTAGGAG TCCAAGGGGT AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC 600 TGGAGCTCCA GGATAGTGGC ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG 660 AGTTCAAAAT AGACATCGTG GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA 720 AAGAGGGGGA ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG 780 GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT 840 TTGACCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA 900 TGGGCAAGAA GCTCCCGCTC CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT 960 CTGGAAACCT CACCCTGGCC CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC 1020 TGGTGGTGAT GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA 1080 CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC 1140 GGGAGAAGGC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG 1200 ACTCGGGACA GGTCCTGCTG GAATCCAACA TCAAGGTTCT GCCCACATGG TCCACCCCGG 1260 TGCAGCCAAT GGCCTCTAGA CTGCTGGACG AGAACAGCAC GCTGATCCCC ATCGCTGTGG 1320 GTGGTGCCCT GGCGGGGCTG GTCCTCATCG TCCTCATCGC CTACCTCGTC GGCAGGAAGA 1380 GGAGTCACGC AGGCTACCAG ACTATCTAGC CTGGTGTCGA C 1421

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1415 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapien (F) TISSUE TYPE: epithelial

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..6

(D) OTHER INFORMATION: /note= "Restriction site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1275?.1280

(D) OTHER INFORMATION: /note= "Restriction site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1410..1415

(D) OTHER INFORMATION: /note= "Restriction site" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAATTCAAGC CCAGAGCCCT GCCATTTCTG TGGGCTCAGG TCCCTACTGC TCAGCCCCTT 60 CCTCCCTCGG CAAGGCCACA ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC 120 TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG 180 GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA 240 AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT 300 CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG GGACCAAGGA AACTTCCCCC 360 TGATCATCAA GAATCTTAAG ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC 420 AGAAGGAGGA GGTGCAATTG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC 480 TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC 540 AATGTAGGAG TCCAAGGGGT AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC 600 TGGAGCTCCA GGATAGTGGC ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG 660 AGTTCAAAAT AGACATCGTG GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA 720 AAGAGGGGGA ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG 780 GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT 840 TTGACCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA 900 TGGGCAAGAA GCTCCCGCTC CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT 960 CTGGAAACCT CACCCTGGCC CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC 1020 TGGTGGTGAT GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA 1080 CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC 1140 GGGAGAAGGC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG 1200 ACTCGGGACA GGTCCTGCTG GAATCCAACA TCAAGGTTCT GCCCACATGG TCCACCCCGG 1260 TGCAGCCAAT GGCCTCTAGA AGTGCAGATG ACGACAACTT CCTTGTGCCC ATAGCGGTGG 1320 GAGCTGCCTT GGCAGGAGTA CTTATTCTAG TGTTGCTGGC TTATTTTATT GGTCTCAAGC 1380 ACCATCATGC TGGATATGAG CAATTTTAGG TCGAC 1415 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1..6

(D) OTHER INFORMATION: /note= "ER retention signal"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Ser Glu Lys Asp Glu Leu 1 5

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..23

(D) OTHER INFORMATION: /note= "PCR Primer P-l used in construction of sCD4-PCaD" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GAATTCAAGC CCAGAGCCCT GCC

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..23

(D) OTHER INFORMATION: /note= "PCR Primer P-2 used in construction of sCD4-PCaD"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TCTAGAGGCC ATTGGCTGCA CCG

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..23 (D) OTHER INFORMATION: /note= "PCR Primer P-3 used in construction of sCD4-PCaD"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TCTAGACTCG TCAGGATCCC GCTG

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..22

(D) OTHER INFORMATION: /note= "PCR Primer P-4 used in construction of sCD4-PCaD"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GTCGACCTAG AGGCGGGCAG CC

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..25

(D) OTHER INFORMATION: /note= "PCR Primer P-l used in construction of SCD4-HAP"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TCTAGACAGC TGGCAAGCGG TCCTG

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..24

(D) OTHER INFORMATION: /note= "PCR Primer P-2 used in construction of SCD4-HAP"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GTCGACTCAG GCGTGGTCCT CCCC

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..26

(D) OTHER INFORMATION: /note= "PCR Primer P-l used in construction of SCD4-L1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TCTAGACTGC TGGACGAGAA CAGCAC

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..28

(D) OTHER INFORMATION: /note= "PCR Primer P-2 used in construction of SCD4-L1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GTCGACACCA GGCTAGATAG TCTGGTAG

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..27

(D) OTHER INFORMATION: /note= "PCR Primer P-l used in construction of SCD4-L2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TCTAGAAGTG CAGATGACGA CAACTTC

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..30

(D) OTHER INFORMATION: /note= "PCR Primer P-2 used in construction of SCD4-L2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GTCGACCTAA AATTGCTCAT ATCCAGCATG

Claims

We claim:

1. A fusion protein comprising: a protein which binds to a retroviral envelope protein and a protein or domain of die protein which targets the fusion protein to a lysosome by causing the protein to be delivered to a lysosome when expressed witiύn a mammalian cell.

2. The protein of claim 1 wherein die binding protein is soluble CD4.

3. The protein of claim 1 wherein the binding protein is selected from die group consisting of sCD4 domain Dl and sCD4 combined domains D1-D2.

4. The protein of claim 1 wherein die targeting protein is selected from the group consisting of procathepsin D (PCaD), human lysosomal membrane protein lamp-1, human lysosomal membrane protein lamp-2, acid phosphatase, and portions thereof targeting the protein to a lysosome.

5. The protein of claim 1 wherein the targeting protein is a lysosomal proenzyme which contains a structural marker for lysosomal targeting.

6. The protein of claim 5 selected from the group consisting of pro-cad epsin D, α-N-Acetylgalactosaminidase, Glycosylasparaginase, Glucocerebrosidase, Procatiiepsin L, Procatiiepsin B, and Procatiiepsin E.

7. The protein of claim 1 wherein the targeting protein is taken from part of tiie lysosomal membrane proteins, lamp-1 (LI), lamp-2 (L2), and lysosomal acid phosphatase (HAP).

8. The protein of claim 1 wherein tiiere are multiple targeting proteins.

9. The protein of claim 8 wherein the fusion protein is selected from the group consisting of sCD4-PCaD-HAP and sCD4-PCaD-PCaD.

10. The protein of claim 1 wherein die targeting protein and the binding protein are separated by a peptide sequence.

11. A gene encoding a fusion protein comprising: a protein which binds to a retroviral envelope protein and a protein or domain of die protein which targets die fusion protein to a lysosome by causing die protein to be delivered to a lysosome when expressed within a mammalian cell.

12. The gene of claim 11 encoding a protein selected from die group consisting of sCD4-PCaD, sCD4-HAP, sCD4-Ll, and sCD4-L2.

13. The gene of claim 11 encoding a fusion protein wherein the binding protein is soluble CD4..

14. The gene of claim 11 encoding a fusion protein wherein tiie binding protein is selected from the group consisting of sCD4 domain Dl and sCD4 combined domains D1-D2.

15. The gene of claim 11 encoding a fusion protein wherein die targeting protein is selected from die group consisting of procatiiepsin D (PCaD), human lysosomal membrane protein lamp-1, human lysosomal membrane protein lamp-2, acid phosphatase, and portions thereof targeting the protein to a lysosome.

16. The gene of claim 11 encoding a fusion protein wherein die targeting protein is a lysosomal proenzyme which contains a structural marker for lysosomal targeting.

17. The gene of claim 11 encoding a fusion protein selected from die group consisting of pro-cathepsin D, o;-N-Acetylgalactosaminidase, Glycosylasparaginase, Glucocerebrosidase, Procatiiepsin L, Procathepsin B, and Procathepsin E.

18. The gene of claim 10 encoding a fusion protein wherein die targeting protein is taken from part of the lysosomal membrane proteins, lamp-1 (LI), lamp-2 (L2), and lysosomal acid phosphatase (HAP).

19. The gene of claim 10 encoding a fusion protein wherein tiiere are multiple targeting proteins.

20. The gene of claim 10 encoding a fusion protein wherein die fusion protein is selected from die group consisting of sCD4-PCaD-HAP and sCD4-PCaD-PCaD.

21. The gene of claim 11 further comprising a vector.

22. A metiiod for treating a viral disease comprising introducing into cells tiiat are infected or exposed to a retrovirus a gene encoding a fusion protein comprising: a protein which binds to a retroviral envelope protein and a protein or domain of die protein which targets die fusion protein to a lysosome by causing die protein to be delivered to a lysosome when expressed within a mammalian cell.

23. The method of claim 22 wherein die virus is human immunodeficiency virus.

24. The metiiod of claim 22 wherein die gene is introduced into e cells widiin a viral vector.

25. The metiiod of claim 22 wherein the gene encodes a protein selected from tiie group consisting of sCD4-PCaD, sCD4-HAP, sCD4- Ll, and sCD4-L2.

26. The metiiod of claim 22 wherein the gene encodes a fusion protein wherein die binding protein is selected from die group consisting of sCD4, sCD4 domain Dl and sCD4 combined domains D1-D2.

27. The metiiod of claim 22 wherein the gene encodes a fusion protein wherein the targeting protein is selected from the group consisting of procathepsin D (PCaD), human lysosomal membrane protein lamp-1, human lysosomal membrane protein lamp-2, acid phosphatase, pro¬ catiiepsin D, α-N-Acetylgakctosaminidase, Glycosylasparaginase, Glucocerebrosidase, Procathepsin L, Procathepsin B, and Procatiiepsin E, and portions thereof targeting the protein to a lysosome.

28. The metiiod of claim 22 wherein die gene encodes a fusion protein including multiple targeting proteins.