Patent Application: US-43963607-A

Abstract:
a hybrid adenovirus semliki forest virus vector includes 3 ′ and 5 ′ inverted terminal repeat of adenovirus , the packaging signal of adenovirus , the structural genes encoding the adenovirus hexon and penton proteins , fiber and knob proteins and that may be deleted in the e4 region , e2 region or in the both the e2 and e4 regions . the adenovirus vector may not require a helper virus coinfection for propagation in producer cell lines . a hybrid vector includes a eukaryotic promoter controlling expression of the 42s genome of sfv comprising the nonstructural genes 1 - 4 or two point mutations thereof , and the therapeutic mrna , in the cytoplasm . in use , the hybrid vector further comprises cdna encoding for microrna and hairpin loops of short interfering rna or cdna encoding for double - stranded rna .

Description:
1 . in order to construct this hybrid virus we have used the adeasy system ( he et al , 1998 ) for the generation of ad vectors . in this system the sfv genome is first cloned into a shuttle plasmid that will be later used to fuse with the ad genome and produce hybrid viral vectors in a complementing cell line ( fig3 ). the sfv ( nsp1 - 4 ) is cloned in this shuttle plasmid in the context of an rna polymerase ii - based expression cassette and contains a tetracyclin - inducible promoter and a sv40 polyadenylation signal : use of the tetracycline controllable expression systems ( the “ tet technology ”) is covered by a series of patents including u . s . pat . nos . 5 , 464 , 758 and 5 , 814 , 618 , which are proprietary to tet systems holding gmbh & amp ; co kg . 2 . the therapeutic sirna is then cloned into the multiple cloning site present immediately after the sfv sgp . 3 . the hybrid shuttle plasmid is then recombined with the ad backbone by their co - transformation into a special strain of bacteria ( he et al , 1998 ) and positive recombinants are selected for further analysis . regulon has used three different ad backbones , each with the standard e1 and e3 deletions for replication incompetence , but which differ by the introduction of an extra deletion at the e2 and / or e4 region . for propagation , e2 - deleted viruses are grown in an e2 - complementing cell line ( amalfitano et al , 1997 ), e4 - deleted viruses are grown in 911e4 cells , an e4 - complementing cell line and e2 / e4 - deleted viruses are grown in a proprietary e2 / e4 complimentary cell line . 4 . high titre stocks of hybrid adsfv vectors expressing therapeutic genes or sirna are prepared and used for increased delivery to the pathologic site , for instance by targeting using specially modified liposomes with tumour / specific peptides . the sequence of each hybrid adenoviral - sfv construct is shown at the end of the present description . the present invention will now be further described with reference to the following examples and drawings which are present for the purposes of illustration only and are not to be construed as being limiting on the invention . fig1 shows the essential elements of the hybrid adenoviral - sfv vector fig3 shows a schematic diagram of hybrid shuttle plasmid at the end of the present description is showed a sequence of hybrid ad - sfv vector containing e1 , e2 and e3 deletions from the adenovirus backbone . the examples describe the use of a hybrid adenoviral - sfv vector for delivery of rna interference constructs against the following targets : 1 . cyclin family of proteins , e . g . cyclin a , b , c , d and e : these are proteins that regulate the cell cycle . disruption of these functions prevents cell division 2 . essential metabolic enzymes , e . g . atpases or enzymes involved in glycolysis and the mitochondrial membrane electron transport chain : these enzymes regulate the essential energy metabolism of the cell . disruption of these functions disrupts cell viability . 3 . p53 mutants : specifically knock down p53 mutants and reexpression of wild type p53 will result in apoptosis only in cancer cells . 4 . aberrant signal transduction molecules e . g . activated tyrosine kinases and tyrosine kinase receptors , egfr , ras , raf , c - myc : these are oncoproteins that drive uncontrolled proliferation of the cell . disruption of these proteins will reduce cell division and promote death of the cell . 5 . genes involved in dna replication , e . g . dna polymerase family of enzymes — alpha , beta , gamma and delta , dna ligases and topoisomerases : these are proteins that control dna replication and repair . disruption of these proteins will prevent cell division and result in cell death . 6 . cdna encoding for microrna ( mirna ) and hairpin loops of short interfering rna or dsrna is directed against drug resistance genes in order to convert drug - resistant tumors to chemotherapy - sensitive . the examples also describes the use of the hybrid adenoviral - sfv vector for the delivery of the following genes for cancer and other diseases ( as indicated ): 1 . tnf - alpha and interferon - gamma for cancer immunotherapy 2 . wild type p53 to induce cancer cell - specific cell death . the ability of p53 to suppress neoplastic growth is lost by mutations on p53 that result in loss of its ability to bind to dna or to interact with other transcription protein factors . mutant p53 can transactivate genes that up - regulate cellular growth such as pcna , egfr , multiple drug resistance ( mdr1 ), and human hsp70 in vivo supporting the idea for an oncogene function of the mutant p53 protein ( for references see boulikas , 1998 , gtmb vol 1 , p54 ). 3 . wild type p53 mutagenized at 2 - 3 nucleotides to abort the pax5 suppressive site and simultaneous insertion of the pax5 cdna whose expression product would suppress the endogenous mutated p53 . effective suppression of tumor growth with p53 vectors could be achieved by the simultaneous transfer of wt p53 plus pax5 to cancer cells ; pax5 is a well established suppressor of the p53 gene ; its effect is exerted via a direct interaction of pax5 with a control element in the first exon of the p53 gene ( stuart et al , 1995 ). pax5 is an homeotic protein , controlling the formation of body structures during development ; pax5 is expressed in early embryo stages to keep the levels of p53 low and allow rapid proliferation of embryonic tissues . simultaneous transfer to solid tumors of a pax5 and p53 genes in the same expression vector but with the wt p53 mutagenized at 2 - 3 nucleotides to abort the pax5 suppressive site is a strategy previously patented to effectively suppress tumor cell proliferation ( boulikas , u . s . pat . no . 6 , 030 , 956 ; issued feb . 29 , 2000 ). 4 . trail to induce rapid programmed cell death . 5 . cip - 1 / waf - 1 / p21 , gadd45 , cyclin g , mdm2 , pcna , muscle creatine kinase mck , egfr , bax , and thrombospondin - 1 . expression of all these genes are upregulated by p53 protein and their upregulation can be applied to specific tumours to suppress tumour cell proliferation ( for references see boulikas , 1998 , gtmb vol 1 , p52 ). the cdnas . gadd45 inhibits cell cycle progression . p21 / cip1 / waf1 and gadd45 interact with pcna to inhibit its association with dna polymerase 8 thus causing arrest in dna replication . mdm2 acts as a feedback loop for the biological functions of p53 apparently to moderate the g1 / s arrest or apoptosis triggered by p53 following severe damage to dna . mdm2 protein associates with p53 causing p53 inactivation by preventing its sequence - specific binding to regulatory targets in dna . elevated levels of mdm2 mimic the effect of t antigen , e1b of adenovirus , e6 of hpv , which also inactivate p53 in a similar manner ; overexpression of mdm2 can block the induction of apoptosis by p53 . the pcna promoter is up - regulated in the presence of moderate amounts of wt p53 ; however , at higher levels of wt p53 the pcna promoter is inhibited whereas tumor - derived p53 mutants activate the pcna promoter . pcna is a protein auxiliary to dna polymerase δ . 6 . hsv - tk , cd , dck , nitroreductase and pnp . these encode prokaryotic or viral enzymes able to convert nontoxic prodrugs into toxic derivatives . the toxic derivative produced in tumor cells which are infected can diffuse to surrounding cells causing their killing even in the absence of infection of these cells , a phenomenon known as “ bystander effect ”. thymidine kinase from hsv uses the 9 -{[ 2 - hydroxy - 1 -( hydroxymethyl )- ethoxy ] methyl } guanine or ganciclovir ( gcv ) and converts to gcv monophosphate for toxicity to cancer cells . cytosine deaminase ( cd ) from e . coli uses 5 - fluorocytosine ( 5fc ) and converts to the toxic agent 5 - fluorouracil ( 5fu ). the e . coli ( deod ) gene encodes the purine nucleoside phosphorylase ( pnp ). the pnp gene product can convert the 6 - methylpurine deoxyribose ( mep - dr ) prodrug into the diffusible , toxic 6 - methylpurine and can become a powerful suicide gene killing infected tumor cells . the method consists of infection of tumors with these adeno - sfv constructs encoding pnp followed by treatment of the patients with mep - dr . purine nucleoside phosphorylase ( pnp ) from e . coli also uses arabinofuranosyl - 2 - fluoroadenine monophosphate ( f - araamp ) commercially known as fludarabine and converts it to a very toxic adenine analog . human deoxycytidine kinase ( dck ) uses cytosine arabinoside ( ara - c ) and converts it to a toxic drug inducing lethal strand breaks in dna . nitroreductase from e . coli uses 5 -( aziridin - 1 - yl )- 2 , 4 - dinitrobenzamide ( cb1954 ) and converts it to a potent dysfunctional alkylating agent which crosslinks dna . 7 . cip - 1 / waf - 1 / p21 , p16 , rb and e1a . introduction of p21 with adenoviral vectors into malignant cells completely suppressed their growth in vivo and also reduced the growth of established pre - existing tumours . one of the most frequent abnormalities in the progression of gliomas is the inactivation of the tumor - suppressor gene p16 , suggesting that loss of p16 is associated with acquisition of malignant characteristics . retinoblastoma ( rb ) protein is a transcription factor involved in the regulation of cell cycle progression genes . the role of rb on cell proliferation and tumor suppression arises ( i ) from its association with e2f , an association disrupted by rb phosphorylation at the g1 / s checkpoint resulting in release of e2f and in the upregulation of a number of genes required for dna replication ; ( ii ) from the direct association of rb protein with a number of viral oncoproteins or key regulatory proteins including e1a of adenovirus , sv40 large t and the human papilloma virus e7 protein . rb also suppresses cell growth by directly repressing transcription of the rrna and trna genes by blocking the activity of rna polymerase i transcription factor ubf . 8 . tgf - β1 , interleukin - 6 ( il - 6 ), il - 2 , interleukin - 1 ( il - 1 ), the tumor necrosis factor - α ( tnf - α ), interferon ( inf )- gamma , granulocyte macrophage colony stimulating factor ( gm - csf ). expression of these genes has pleiotropic effects on various tumors and normal cells and can also be used for cancer immunotherapy as well as against viral infections and other diseases . 9 . transcription factors e2f , rbf - 1 , atf , ap - 1 , sp1 , nf - κb . expression of these genes has pleiotropic effects on various tumors and normal cells and can also be used for triggering apoptosis of tumor cells and in other diseases . 10 . bax , bc1 - 2 , bc1 - xs , bc1 - xl , c - myc , interleukin - 1β converting enzyme ( ice ), poly ( adp - ribose ) polymerase ( parp ). expression of these genes has pleiotropic effects on various tumors and normal cells and can also be used for triggering apoptosis of tumor cells , in autoimmune disease , in ischemic heart disease and in other diseases . 11 . erk1 , erk2 , mek1 , mek2 , mek3 , mek4 , mek6 kinases , ceramide - activated kinase , iκb kinase , raf - 1 , jun n - terminal kinases or jnks , p38 / mpk2 ), mitogen - activated protein kinase ( mapk )/ extracellular signal - regulated kinase ( erk ) kinase kinase 1 ( mekk1 ) kinases . expression of these genes has pleiotropic effects on various tumors and normal cells and can also be used for triggering apoptosis of tumor cells and in other diseases . 12 . adenosine deaminase ( ada ) used for scid ( severe combined immunodeficiency ), bc1 - 2 for cancer , factor viii for hemophilia a , factor ix for hemophilia b , growth hormone ( human ) for increase in growth , hsv - tk for proliferative vitreoretinopathy ( pvr ), il - 1 receptor antagonist ( il - 1ra ) for rheumatoid arthritis ( ra ), ldl receptor for familial hypercholesterolemia ( fh ), nerve growth factor ( ngf ) for alzheimer &# 39 ; s disease and multiple sclerosis , xpd ( ercc2 ) for xeroderma pigmentosum ( xp ), th ( tyrosine hydroxylase ) for parkinson &# 39 ; s disease ( pd ). 13 . cyclin - 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