# Patent

## Title
Adenovirus/alphavirus hybrid vector for the effective administration and expression of therapeutic genes in tumour cells

## Abstract

The present invention relates to a genic expression adenoviral hybrid vector characterized in that it contains at least the following elements, oriented in the direction 5′ to 3′: i. a first chain of adenoviral origin comprising a first inverted terminal repeat (ITR) sequence and a signal sequence for packaging of the adenovirus; ii. a first non-encoding stuffer sequence; iii. a sequence corresponding to a tissue specific promoter; iv. a chain of cDNA derived from an alphavirus, the sequence of which is partly complementary to an alphaviral RNA sequence, comprising at least a sequence encoding for at least one exogenous gene of interest; v. a polyadenylation sequence; and vi. a second adenoviral inverted terminal repeat (ITR) sequence, it preferably relates to an adenoviral hybrid vector comprising as exogenous gene of interest the therapeutic gene of mammalian interleukin IL-12 and even more preferably human interleukin hIL-12; and to the use of the hybrid vector in a process for transferring genetic material to a cell, particularly a tumor cell that preferably expresses alpha-fetoprotein (AFP), and to its use for inducing an immune response against foreign antigens.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is filed under the provisions of 35 U.S.C. §371 and claims the priority of International Patent Application No. PCT/ES2005/000277 filed on 18 May 2005, which in turn claims priority of Application No. P200401219 (España) filed on 20 May 2004.
TECHNICAL FIELD OF THE INVENTION
The present invention refers to genic expression vectors derived from adenoviruses, for the obtainment of therapeutic products.
STATE OF THE ART PRIOR TO THE INVENTION
One of the principal problems facing conventional cancer therapy is the lack of tumor specificity, a situation that often leads to serious side effects and limits the applicable therapeutic dose. Although gene therapy remains highly promising for cancer treatment, it also faces a specific problem: targeting transgenic expression to the tumor site. A number of studies suggest that when viral vectors are administered on an intralesional basis, although transgenic expression is essentially confined to a region adjacent to the needle trajectory, though such expression may also occur in other tissues. Consequently, an important objective in gene therapy applied to cancer is the targeting of therapeutic gene expression to the tumors via specific administration to the neoplastic tissue (“tissue objective”) and/or specific activation (“transcriptional objective”) within the neoplastic tissue, without affecting the healthy cells. The “tissue objective” can be achieved by creating a vector targeted via modifications of the receptor-ligand interactions, thus allowing the infection of cells that express a specific receptor. The “transcriptional objective” can be achieved using a tumor specific promoter to control transgenic expression. Previous studies have used different tumor specific promoters. However, these present an essential limitation in that they do not yield high levels of genic expression, as a result of which the antitumoral activity is limited.
A review of the latest advances in the development of viral vectors for gene therapy can be found in Lundstrom K. “Latest development in viral vectors for gene therapy”; Trends in Biotechnology, 2003, 21:118-122.
Currently used viral vectors include the alphaviruses. Alphaviruses are enveloped viruses that contain a simple positive RNA strand as genome. Expression vectors derived from the alphaviruses Sindbis Virus (SIN), Semliki Forest Virus (SFV), and Venezuelan equine encephalitis (VEE) virus have been designed and developed. The alphavirus vectors are based on the use of self-replicating RNA molecules derived from alphaviral genomes in which the 5′ and 3′ sequences necessary for replication and the replicase (Rep) gene have been maintained, while the genes encoding for the viral structural proteins have been deleted and replaced with a transgene. Following cell transfection of these vectors, Rep is translated and the RNA vector is copied in a negative RNA strand, which will be used as a template for amplification of the RNA vector. Rep can also recognize a subgenomic promoter in the negative RNA strand, from which a smaller subgenomic RNA segment is in turn synthesized that can be translated to produce important levels of heterologous proteins. Alphaviral vectors can be used directly as RNA when transcribed in vitro from a prokaryotic promoter such as SP6 or T7, or as DNA when the replicon sequence is located under an eukaryotic promoter such as CMV. The RNA vector can be packaged in viral particles via its cotransfection in cells, together with one or more “helper” RNAs that encode for the viral structural proteins. Alphaviral vectors possess a series of properties that make them attractive for gene therapy: a very extensive tropism, low immunogenicity, and a high level of heterologous protein expression. Such expression is, however, transient due to the induction of apoptosis in the cells when replication takes place. The document Rayner J. O., Dryga S. A., Kamrud K. I. “Alphavirus vectors and vaccination”; Rev. Med. Virol. 2002; 12 279-296, describes the development of expression vectors based on alphaviruses for use in the field of vaccines.
Another series of viral vectors are based on adenoviruses. There is extensive literature on the use of adenoviruses, which have been developed to overcome some of the inconveniences of gene therapy, and as a source for the creation of expression vectors. A document reporting the latest advances in the field of adenoviral vectors has been published by Volpers C, Kochanek S. “Adenoviral vectors for gene transfer and therapy”; J Gene. Med. 2004; 6: 5164-5171. Adenoviruses have the advantage of affording high transduction efficiency and the capacity to persist in episomal form. However, the expression of adenoviral proteins induces potent immune responses that limit the duration of transgenic expression and induce toxicity in the cells infected with the vector. Gutless adenoviruses have been developed to solve these problems. These gutless adenoviruses have been deprived of all the corresponding adenoviral genes (the only sequences preserved being the two inverted terminal repeat sequences and the packaging signals), consequently, the transduced cells express no adenoviral product and do not induce an immune response to the vector. In short, the elimination of all the adenoviral genes leaves sufficient space to house large expression cassettes and that is why gutless adenoviruses are also called high-capacity adenoviral vectors. A document describing concrete aspects of adenoviral vectors, related to the deletion of all the sequences encoding for viral proteins, has been published by Morsy M A et al. “An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene”; Proc. Natl. Acad. Sci. USA 1998, 95:7866-7871. The document published by Schiedner G et al. “Variables affecting in vivo performance of high-capacity adenovirus vectors”; J. Virol. 2002, 76:1600-1609 describes the use of stuffer DNA in expression vectors based on gutless adenoviruses, showing that the presence of such stuffer DNA is essential for achieving a considerable increase in genic expression, and that, in general, the designing of vectors based on high-capacity (gutless) adenovirus can substantially modify the degree and duration of expression of a gene.
On the other hand, document U.S. Pat. No. 5,981,225 describes a vector for gene transfer based on adenoviruses that comprises inverted terminal repeat (ITR) sequences, at least one packaging signal sequence, and a VAI adenoviral gene and/or VAII adenoviral gene; and comprises a gene foreign to the adenovirus operatively bound to a functional promoter in target cells for adenovirus.
The document U.S. Pat. No. 5,985,846 describes a gene transfer vector comprising inverted terminal repeat (ITR) sequences of adenovirus and recombinant particles of adenovirus containing these sequences.
The document U.S. Pat. No. 6,566,093 describes vectors of cDNA derived from alphaviruses that consist of DNA complementary to at least part of the RNA of an alphavirus, essential for replication of the alphavirus, and heterologous cDNA, for example, cDNA encoding for a desired substance. The latter can be a biologically active protein or polypeptide, as well as an immunogenic or antigenic protein or polypeptide, a therapeutically active protein or polypeptide, or a therapeutically active RNA.
The purpose of the present invention is to improve transgenic expression and the induction of apoptosis in tumor cells mediated by hybrid vectors in vitro and in vivo. An additional objective is to improve the efficiency of tumor therapy in animal models by means of hybrid vectors.
An additional objective is also to develop a method of gene therapy, in particular for the treatment of cancer, by means of the use of hybrid vectors.
The objectives of the present invention are achieved by combining the following in a single vector:
high infective capacity, by using an adenovirus release system,
high transgenic expression and the induction of apoptosis by using a vector derived from an alphavirus, such as SFV, and
tumor specificity by using a tumor specific promoter.
DESCRIPTION OF THE INVENTION
The present invention firstly relates to a genic expression adenoviral hybrid vector characterized in that it comprises at least the following elements, oriented in the direction 5′ to 3′:

    
    
        i. a first chain of adenoviral origin comprising a first inverted terminal repeat (ITR) sequence and a signal sequence for packaging of the adenovirus;
        ii. a first non-encoding stuffer sequence;
        iii. a sequence corresponding to a tissue specific promoter;
        iv. a chain of cDNA derived from an alphavirus, the sequence of which is partly complementary to an alphaviral RNA, comprising at least a sequence encoding for at least one exogenous gene of interest;
        v. a polyadenylation sequence; and
        vi. a second adenoviral inverted terminal repeat (ITR) sequence.
    
    


More specifically, the present invention relates to the construction of an adenoviral hybrid vector comprising as element iv a chain of cDNA derived from an alphavirus corresponding to the sequence of an SFV recombinant replicon, under the transcriptional control of a tumor specific promoter (element iii), which is the promoter of alpha-fetoprotein (AFP). In this construct, a transgene can be inserted in the SFV replicon, directed by the subgenomic promoter of SFV. Following infection of the tumor cells with this hybrid vector, the mRNA of the SFV replicon is transcribed from the tumor specific promoter, and the non-structural proteins—nsPs—which constitute the SFV replicase gene are translated from said mRNA of the SFV replicon. These nsPs proteins—viral replicase—initiate replication of the mRNA of the SFV replicon, to generate the subgenomic RNA of SFV. Consequently, the transgene can be expressed at a high level from the subgenomic RNA of SFV. This global viral replication process will give rise to the production of apoptosis in the infected cells. In the event that these hybrid infect non-tumor cells, the mRNA of the SFV replicon will not be transcribed from the tumor specific promoter, which will not be active in these cells. Thus, there will be no expression of the transgene, and apoptosis will not occur in normal cells infected with the hybrid vector.
The present invention further relates to a method for obtaining said adenoviral hybrid vector, comprising the assembly of elements i. to vi. of the adenoviral hybrid vector defined above by means of genetic engineering techniques.
The present invention also relates to the use of the mentioned vector hybrid for transferring genetic material to a cell, and more particularly for introducing and expressing foreign genes in eukaryotic cells that may be target cells for adenovirus.
The transfer of genetic material preferably results in the induction of an immune response against foreign antigens in said cell.
The present invention also relates to a pharmaceutical composition comprising said adenovirus-alphavirus hybrid vector and its use in the therapeutic treatment of cancer, comprising the administration of said pharmaceutical composition to a subject.
The present invention also relates to a method for cancer treatment by means of the use of the previously defined hybrid vector, comprising the administration of said hybrid vector to a subject.
The present invention relates to a genic expression adenoviral hybrid vector characterized in that it comprises at least the following elements, oriented in the direction 5′ to 3′:

    
    
        i. a first chain of adenoviral origin comprising a first inverted terminal repeat (ITR) sequence and a signal sequence for packaging of the adenovirus;
        ii. a first non-encoding stuffer sequence;
        iii. a sequence corresponding to a tissue specific promoter;
        iv. a chain of cDNA derived from an alphavirus, the sequence of which is partly complementary to an alphaviral RNA sequence, comprising at least a sequence encoding for at least one exogenous gene of interest;
        v. a polyadenylation sequence; and
        vi. a second adenoviral inverted terminal repeat (ITR) sequence.
    
    


The nature of element i. in the alphavirus-adenovirus hybrid vector of the present invention, i.e., the nature of the chain of adenoviral origin comprising a first inverted terminal repeat (ITR) sequence and a signal sequence for packaging (ψ) of the adenovirus, is not a critical aspect for the present invention, and may originate from any adenovirus serotype. Said serotypes are well known in the technique and include for example Ad12 (subgenus A), Ad3 and Ad7 (subgenus B), Ad2 and Ad5 (subgenus C), Ad8 (subgenus D), Ad4 (subgenus E), Ad40 (subgenus F), and other known non-human adenoviruses that may originate from species such as pigs, sheep, cows and birds. Therefore, this first inverted terminal repeat sequence which may contain approximately between 100 and 500 bp in length, can vary according to the adenovirus serotype used. In the same way, the signal sequence for packaging of the adenovirus can vary according to the adenovirus serotype employed.
According to a preferred particular embodiment, said genic expression adenoviral vector comprises an element i. having SEQ ID No 1, or any other sequence having sufficient homology with SEQ ID No 1 for performing the same function.
The nature of element ii. in the adenoviral hybrid vector of the present invention is not a critical aspect of the same. Said element ii., the function of which is to increase the total size of the construct, can be any non-encoding stuffer sequence. Said sequence is preferably a human non-encoding sequence. Even more preferably, this non-encoding stuffer sequence is the intron region of human genomic hypoxanthine phosphoribosyltransferase (HPRT).
Preferably, the defined adenoviral hybrid vector also comprises an element vii., which is a second non-encoding stuffer sequence, located between element v. and element vi. defined above.
The nature of element iii. in the adenoviral hybrid vector of the present invention is not a critical aspect thereof. The tissue specific promoter iii. is preferably a tumor specific promoter. Examples of tumor specific promoters include the promoters AFP, telomerase TERT, PAP (pancreatic associated protein), E2F and HIF.
According to a preferred particular embodiment of the invention, the tumor specific promoter has the sequence SEQ ID No 7 corresponding to the AFP promoter/enhancer, (AFP p+e), or the sequence SEQ ID No 15 corresponding to telomerase TERT, or any other sequence having sufficient homology with the sequence SEQ ID No 7 or with the sequence SEQ ID No 15, for performing the same function, respectively.
The nature of element iv. in the adenoviral hybrid vector of the present invention is not a critical aspect thereof. The alphaviral sequences of element iv. are preferably derived from the Semliki Forest Virus (SFV). However, it would be possible to use other alphaviral sequences derived from any of the species belonging to the Togaviridae family, for example SIN, RRV and VEE.
Said chain iv. of cDNA derived from an alphavirus, the sequence of which is partly complementary to alphaviral RNA, preferably comprises (in addition to a sequence encoding for at least one exogenous gene of interest):

    
    
        a) a 5′ sequence necessary for replication of the alphavirus,
        b) a sequence encoding for the non-structural proteins required for replication of the alphaviral RNA,
        c) at least one subgenomic promoter of the alphavirus, and
        d) a 3′ sequence necessary for replication of the alphavirus;
    
    


Element iv. preferably forms a replicon functionally controlled by the promoter iii., and where in turn the alphaviral subgenomic promoter comprised in iv. c) functionally controls the expression of the exogenous gene of interest.
According to a preferred particular embodiment, the sequences a) to c) of element iv. as a whole have a sequence selected from SEQ ID No 3 (SFV 5′-rep-Psg) or any other sequence having sufficient homology with SEQ ID No 3 for performing the same function, and SEQ ID No 4 (SFV 5′-rep-Psg-enh) or any other sequence having sufficient homology with SEQ ID No 4 for performing the same function.
According to an especially preferred particular embodiment, element iv. d) has the sequence SEQ ID No 5 (SFV3′), or any other sequence having sufficient homology with SEQ ID No 5 for performing the same function.
In element iv. of the alphavirus-adenovirus hybrid vector of the present invention, the exogenous gene of interest is preferably a therapeutic gene or a reporter gene, or a combination of both. Without being considered as limiting, the therapeutic gene is preferably selected from mammalian interleukin IL-12, colony stimulating factor GMCSF, alpha-interferon, and herpes simplex virus thymidine kinase (tk).
The exogenous gene of interest in element iv. may further be a reporter gene. Without being considered as limiting, the reporter gene can be selected from LacZ, Luciferase, tk and GFP.
The therapeutic gene is especially preferably mammalian interleukin IL-12, and even more preferably the therapeutic gene is human interleukin, hIL-12.
The genic expression adenoviral hybrid vector can include in element iv in series one or several subsets of (subgenomic promoter+exogenous gene of interest).
The nature of element v. in the adenoviral hybrid vector of the present invention is not a critical aspect thereof. Element v. is preferably a polyadenylation sequence of SV40. Said polyadenylation sequence of SV40 is especially preferable sequence SEQ ID No 6, or any other sequence having sufficient homology with sequence SEQ ID No 6 for performing the same function.
The nature of element vi. in the adenoviral hybrid vector of the present invention, is not a critical aspect thereof. According to a preferred embodiment, said genic expression adenoviral vector comprises an inverted terminal repeat (ITR) sequence as element vi., which has sequence SEQ ID No 2, or any other sequence having sufficient homology with SEQ ID No 2 for being able to perform the same function.
The nature of element vii. in the adenoviral hybrid vector of the present invention is not a critical aspect thereof. The second non-encoding stuffer sequence can be any such sequence. It is preferably a human non-encoding sequence, and especially preferably a sequence from human cosmid C346.
The genic expression adenoviral hybrid vector of the present invention can have a variable length, and preferably has a length comprised between 27 and 38 kilobases.
According to a preferred particular embodiment, the adenoviral hybrid vector comprises ITR 5′ as a first inverted terminal repeat sequence; HPRT, the intron region of human genomic hypoxanthine phosphoribosyltransferase as a first stuffer sequence; AFP (p+e), a tumor specific promoter; an SFV replicon sequence containing mIL-12, interleukin-12 from mice; SV40 PolyA, a polyadenylation sequence of SV40; C346, human genomic cosmid C346 as a second stuffer sequence, and ITR 3′ as a second inverted terminal repeat sequence.
According to an additional preferred particular embodiment, the adenoviral hybrid vector comprises ITR 5′ as a first inverted terminal repeat sequence; HPRT, the intron region of human genomic hypoxanthine phosphoribosyltransferase as a first stuffer sequence; AFP (p+e), a tumor specific promoter; an SFV replicon sequence containing LacZ; SV40 PolyA, a polyadenylation sequence of SV40; C346, human genomic cosmid C346 as a second stuffer sequence, and ITR 3 as a second inverted terminal repeat sequence.
According to a particularly preferred additional embodiment, the adenoviral hybrid vector comprises ITR 5′ as a first inverted terminal repeat sequence; HPRT, the intron region of human genomic hypoxanthine phosphoribosyltransferase as a first stuffer sequence; AFP (p+e), a tumor specific promoter; an SFV replicon sequence containing hIL-12, human interleukin-12; SV40 PolyA, a polyadenylation sequence of SV40; C346, human genomic cosmid C346 as a second stuffer sequence, and ITR 3′ as a second inverted terminal repeat sequence.
According to a preferred particular embodiment of the present invention, the genic expression adenoviral hybrid vector has sequence SEQ ID No 8, or any other sequence having sufficient homology with SEQ ID No 8 for performing the same function.
According to an additional preferred particular embodiment of the present invention, the genic expression adenoviral hybrid vector has sequence SEQ ID No 9, or any other sequence having sufficient homology with SEQ ID No 9 for performing the same function.
According to an additional preferred particular embodiment of the present invention, the genic expression adenoviral hybrid vector has sequence SEQ ID No 10, or any other sequence having sufficient homology with SEQ ID No 10 for performing the same function.
The present invention further relates to a method for obtaining said adenoviral hybrid vector, comprising the assembly of elements i. to vi., or i. to vii., of the adenoviral hybrid vector defined above by means of genetic engineering techniques.
The present invention also relates to the use of said hybrid vector for transferring genetic material to a cell, and more particularly for introducing and expressing foreign genes in eukaryotic cells that may be target cells for adenoviruses. Said use comprises administering said hybrid vector to a subject.
The infection of tumor cells with an adenoviral hybrid vector according to the invention, results in the transcription of the mRNA of the SFV alphavirus replicon from the tumor specific promoter, therefore the Rep gene will be translated and the RNA of SFV will be amplified. Rep also produces a subgenomic RNA of SFV, from which the therapeutic or reporter gene will be expressed at high levels. The product of the therapeutic gene secreted by the infected cells will activate the immunocytes at the infection site. The replication of SFV will further induce apoptosis of the infected cells, leading to the release of tumor antigens from the apoptotic cells; which can be captured by antigen-presenting cells (APCs), thereby activating the immune response against the tumor. However, if this hybrid vector infects non-tumor cells, the mRNA of the SFV replicon will not be transcribed, and therefore no transgenic expression or apoptosis will occur.
The tumor cells are preferably infected with an adenoviral hybrid vector according to the invention, such that the mRNA of the SFV replicon is transcribed from the tumor specific promoter AFP, therefore the Rep gene will be translated and the RNA of SFV will be amplified. Rep will also produce a subgenomic RNA of SFV, from which mIL-12 or hIL-12 will be expressed at high levels. mIL-12 or hIL-12 secreted by infected cells will activate immunocytes at the infection site. The replication of SFV will further induce apoptosis in the infected cells, leading to the release of tumor antigens by the apoptotic cells, which can be captured by antigen-presenting cells (APCs), thereby activating the immune response against the tumor. However, if this hybrid vector infects non-tumor cells, the mRNA of the SFV replicon will not be transcribed, and therefore no transgenic expression or apoptosis will occur.
An additional objective of the present invention is the use of an adenoviral hybrid vector defined previously in a process for transferring genetic material to a cell, preferably a tumor cell, and which comprises administering said hybrid vector to a subject. Even more preferably, said cell is a tumor cell that expresses AFP.
An additional objective of the present invention is the use of a defined adenoviral hybrid vector for the preparation of an effective medicament in the treatment of tumors, and its use for inducing an immune response against foreign antigens. Said use comprises administering said medicament to a subject.
An additional objective of the present invention is a pharmaceutical composition comprising at least an adenoviral hybrid vector defined according to the present invention, and the use thereof in a process for the treatment of tumors, or for inducing an immune response to foreign antigens.
Said pharmaceutical composition preferably comprises an adenoviral hybrid vector according to the present invention, in which the exogenous gene of interest is the mammalian interleukin, IL-12, preferably human interleukin hIL-12. Said use comprises administering the pharmaceutical composition comprising said hybrid vector to a subject.
The present invention also relates to a method for the treatment of cancer by means of the use of the hybrid vector according to the present invention, said method comprising the administration of said hybrid vector to a subject.
Therefore, according to preferred embodiments of the present invention, AFP (p+e) has been selected as a tumor specific promoter, two adenoviral hybrid vectors have been constructed in which the SFV replicon is controlled by the AFP promoter, and the reporter gene LacZ and the therapeutic gene IL-12 are inserted under the control of the SFV subgenomic promoter, respectively—Ad/AFP-SFV-LacZ and Ad/AFP-SFV-mIL-12; and two gutless adenoviral vectors carrying Lacz and IL-12 from mice directly controlled by the AFP promoter—Ad/AFP-LacZ and Ad/AFP-mIL-12—have been prepared as control vectors. It has been shown that the hybrid vector of the present invention works more effectively than the control vectors used to date.
According to the present invention, it has been shown that the vector Ad/AFP-SFV-mIL-12 can be a useful vector in the therapy of HCC (hepatocarcinoma) tumors expressing AFP.
It has also been shown that the use of other tumor promoters such as telomerase promoter, TERT, which is widely activated in most malignant tumors for controlling SFV can convert the use of a hybrid vector such as that of the present invention into a general strategy for the treatment of all types of cancer.
Furthermore and advantageously, the hybrid vector of the present invention specifically works with tumor cells and destroys tumor cells without the need to incorporate a therapeutic gene. Additionally, it is shown that the hybrid vector of the present invention advantageously induces a potent anti-tumor activity when it includes a therapeutic gene such as IL-12.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 (A): shows the diagram of a hybrid vector according to an embodiment of the invention, Ad-SFV, containing a gutless adenovirus sequence in which an SFV replicon has been inserted under the control of the AFP promoter/enhancer (AFP (p+e)), and containing the heterologous mIL-12 gene, which is placed under the control of the SFV subgenomic promoter (Pr sg).
FIG. 1 (B) shows the anti-tumor activity of the hybrid vector according to the invention: following infection of the HCC tumor cells with this hybrid vector (right), thanks to the presence in the hybrid vector of the SFV replicon comprising mIL-12, high-level expression of mIL-12 occurs, which will activate the immunocytes at the infection site. The replication of SFV will further induce apoptosis of the infected cells. However, if this hybrid vector infects non-tumor cells, the mRNA of the SFV replicon will not be transcribed, and consequently there will be no transgenic expression or apoptosis. In this Figure:
ITR, adenoviral inverted terminal repeat sequences;
ψ, adenoviral packaging signal;
HPRT and C346, sequences of stuffer DNA from the intron region of human genomic hypoxanthine phosphoribosyltransferase or from the human cosmid C346, respectively;
PolyA, polyadenylation signal (for example, of SV40);
APCs, antigen-presenting cells.
FIG. 2 shows the structure of the gutless adenoviral hybrid vectors and gutless adenoviral vectors. AFP-SFV-LacZ and AFP-SFV-mIL-12 are hybrid adenovirus vectors, in which the SFV replicon sequence is under the control of the AFP promoter/enhancer (AFP(p+e)), and the heterologous genes LacZ or mIL-12 have been cloned under the control of the SFV subgenomic promoter (Pr sg), respectively. AFP-lacZ and AFP-mIL-12 are adenoviral vectors containing LacZ or mIL-12 directly controlled by AFP (p+e). SFV nsp1-4, non-structural proteins of SFV.
FIG. 3 shows the specific expression of mIL-12 in vitro in hepatocarcinoma cells expressing AFP, HCC (A), and in cells not derived from HCC (B) following infection with the hybrid vectors of Ad-SFV: AFP-mIL-12 (AFP-12), AFP-SFV-mIL-12 (AFP-SFV-12), or with the control vector AdCMVmIL-12 (CMV-12). Different multiplicities of infection “moi” were tested (10, 100 and 1000). Hep3B, Huh-7, HepG2 and PLC/PRF/5: HCC cell lines; Hela, A549, MHC1, SK-Hep-1 and Clon 9: lines not derived from HCC. FIG. 3B only shows the expression corresponding to moi 1000.
FIG. 4 shows the specific expression of β-gal in 4 HCC cell lines (Hep3B, Huh-7, HepG2 and PLC/PRF/5) following in vitro infection with the hybrid vector AFP-SFV-lacZ or with the control vector AFP-LacZ at different “moi” (10, 100, or 1000).
FIG. 5 shows the analysis of the expression of β-gal in HCC cell lines infected with AFP-LacZ and AFP-SFV-LacZ. Microphotographs of cells infected with AFP-LacZ (A-C) or AFP-SFV-LacZ (D-F) and stained with X-Gal. A and D, Hep3B; B and E, Huh7; C and F, HepG2.
FIG. 6 shows the expression kinetics of IL-12 in HCC cell lines Hep3B (A) and Huh-7 (B), infected in vitro with adenoviral vectors AFP-mIL-12 (AFP-12) or AFP-SFV-mIL-12 (AFP-SFV-12), at a “moi” of 1000.
FIG. 7 shows the induction of cell death following the in vitro infection of HCC cell lines—Hep3B (A) and McA-RH7777 (B)—with vectors AFP-IL-12 (AFP-12), AFP-SFV-IL-12 (AFP-SFV-12), AFP-LacZ, AFP-SFV-LacZ, or control vector Ad/CMVmIL-12 (CMV-12). Cell survival is shown as the percentage of live cells in infected wells compared with the live cells in uninfected control wells.
FIG. 8 shows the expression of SFV Rep in HCC cells—Hep3B (A-D) and Huh-7 (E and F)—following infection with vectors AFP-mIL-12 (A and B) or AFP-SFV-mIL-12 (C-F), at a “moi” of 1000. Two days after infection the cells were fixed and analyzed by immunofluorescence with a specific antibody for Rep. The cells expressing Rep were visualized under a fluorescence microscope with an FITC filter (A, C, and E), while the nuclei stained with DAPI in all cells were visualized with a UV filter (B, D, and F).
FIG. 9 shows the gene transfer with gutless hybrid vectors in vivo, (A-D), efficiency of the gene transfer and induction of apoptosis in Huh-7 tumors. Human Huh-7 tumors established in nude immunodeficient mice were treated via intratumor injection with the vectors AFP-LacZ (n=4) or AFP-SFV-LacZ (n=4), at 1×1010 viral particles/animal. Three days after the administration of the virus, the mice were sacrificed and sections of the tumor were analyzed to study the transgene expression by staining with X-Gal (A-B) or to study the induction of apoptosis by means of TUNEL (C-D). A, C; Microphotographs or tumors that received AFP-LacZ. B, D. Microphotographs of tumors that received AFP-SFV-LacZ. (E-G), Specificity of the gene expression with gutless hybrid vectors. Healthy Balb/c mice were injected intravenously with 1×1010 viral particles of the vectors AFP-LacZ (E), AFP-SFV-LacZ (F) or Ad/CMV-LacZ (G). Microphotographs of hepatic tissue sections taken three days after the inoculation and stained with X-Gal are shown.
FIG. 10 shows the treatment of HCC tumors with hybrid vectors. Orthotopic HCC tumors were established via the implantation of McH-RH7777 cells in rat liver. When the tumor reached a size of 7-10 mm in diameter, the animals were treated with 1011 (A-C) or with 2×1011 (D-G) viral particles of AFP-mIL-12, AFP-SFV-mIL-12, or saline solution as control. The size of the tumor was measured on days 15 and 30 following the administration of saline solution (A and E), AFP-mIL-12 (B and F) or AFP-SFV-mIL-12 (C and G). G; Animal survival rate.
FIG. 11 shows the toxicity study in rats inoculated with vectors expressing IL-12. The level of transaminases (GPT, GOLT, and GGTL) (A) or of IL-12 (B) was determined in the serum of rats carrying HCC tumors in liver and which had been inoculated intratumorally with the adenovirus vectors AFP-SFV-IL-12, AFP-SFV-mIL-12, alphavirus vector SFV-IL-12, or with saline solution. The measurement was made on days 4 and 8 after the treatment.
FIG. 12 shows the staining with hematoxylin/eosin of liver sections of rats treated with the adenoviral hybrid vectors. Rats carrying HCC tumors were treated via intratumor injection with saline solution (A), with the adenoviral vectors AFP-IL-12 (B), AFP-SFV-IL-12 (C), or with viral particles of SFV-IL-12 (D). Three days after the treatment the animals were sacrificed, the livers were removed and fixed with formol, and sections were obtained and stained with hematoxylin/eosin. The black arrows indicate areas with eosinophilic hepatocytes.
FIGS. 13A and 13B show restriction maps of plasmids pGL3/AFP and pBS/mIL-12, respectively.
FIGS. 14A and 14B show restriction maps of plasmids pTGC3001 and pTGC3011, respectively.
FIGS. 15A and 15B show restriction maps of plasmids pTGC3012 and pTGC3013, respectively.
FIG. 16 shows the restriction map of plasmid pTGC3014.



INVENTION EMBODIMENTS
Plasmids
pGEM-T “easy” and pCMVb were obtained from Promega, U.S.A., and PBS-SK+ from Stratagene, U.S.A. pSTK120 was kindly donated by Dr. Kochanek (University of Ulm, Germany). pBK-SFV-1 and pBK-SFV-3 have been described by Berglund P. et al. “Enhancing immune responses using suicidal DNA vaccines”. Nature Biotechnology 1998, 16:562-565. pGL3/AFP and pBS/mIL-12 (Yonglian Sun, Cheng Qian, Dacheng Peng and Jesús Prieto. 2000. Gene transfer to liver cancer cells of B7-1 in addition to IL-12 changes immunoeffector mechanisms and suppresses Th1 cytokine production induced by IL-12 alone. Human Gene Therapy 11:127-138) were produced in our laboratory.
For the construction of pGL3/AFP, the regions of the AFP promoter/enhancer (p+e) were obtained via PCR amplification of human genomic DNA. The primers used for amplification of the AFP promoter (AFP pro) were SEQ ID NO. 16: CTCTAGATTTTCTGCCCCAAAGAGCTC and SEQ ID NO. 17: CGGGATCCTGTTATTGGCAGTGGTGGAA. The primers used for amplification of the AFP enhancer were SEQ ID NO. 18: CGGAATTCGCCTGTCATACAGCTAATAA and SEQ ID NO. 19: CTCTAGACTGTCAAATAAGTGG CCTGG. The sequences of the promoter (217 base pairs) and of the enhancer (785 base pairs) were cloned in pGEM-T plasmids. Subsequently, confirmation of the amplified fragments was carried out via sequencing. The AFP promoter was removed from the pGEM-T/AFP-p plasmid by restriction with Xba I/BamHI, and inserted by blunt-end ligation in a pGL3-basic plasmid digested with Sma I. In this way a pGL3/AFP-p plasmid was obtained. The AFP enhancer was removed from the pGEM-T/AFP-e plasmid by restriction with Xba I/Eco RI and was inserted via blunt-end ligation in the pGL3/AFP-p plasmid digested with Nhe I, to finally obtain the pGL3/AFP plasmid.
Cell Lines and Tissue Cultures
The human HCC cell lines Hep3B, PLC/PRF/5, HepG2 and SK-Hep-1, the human cervical epithelial adenocarcinoma cell line HeLa, the human lung carcinoma cell line A549, the human embryonic kidney cell line 293, the rat HCC cell McA-RH7777, MHC1, and Clone 9 of normal rat hepatocytes and the rat HCC cell line Hepa1-6, were obtained from the ATCC. The 293 cells expressing Cre recombinase (293Cre4) were obtained from Merck Research Laboratories. The Hep3B, PLC/PRF/5, HeLa, SK-Hep-1, Clone 9, Huh-7 and Hepa1-6 cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) inactivated by heating and penicillin/streptomycin. The HepG2 and A549 cells were grown in RPMI 1640 medium supplemented with 10% FBS inactivated by heating and penicillin/streptomycin. The McH-RH7777 and MHC1 cells were grown in DMEM medium supplemented with 20% horse serum and 5% FBS. The 293Cre4 cells were grown in DMEM medium supplemented with 10% FBS and 0.4 mg/ml G418.
Animals
Seven-week-old female nude immunodeficient BALB/c mice were obtained from Charles Rivers Laboratories (Barcelona, Spain). Male Buffalo rats aged 4-6 weeks were obtained from CIFA (Animal Installations of the University of Navarra). Mice and rats were housed under the usual conditions in CIFA. The nude mice received an irradiated diet with autoclaved drinking water. Handling of the nude mice was always carried out under a laminar flow chamber. All procedures with animals were carried out in adherence to the standard recommendations and protocols for the care and use of laboratory animals.
Construction of Vectors
Construction of Expression Cassettes AFP-SFV
The 5′ end sequence of SFV (1-292 nt) was amplified by PCR using the pBK-SFV-1 plasmid (containing the full sequence of the SFV replicon) as a template. Primer 1 contained an Spe I restriction site at the 5′ end (underlined) followed by 50 nt of the AFP promoter sequence and the first 20 nt of the SFV sequence (in italics): SEQ ID NO. 20: 5′-ACT AGT TAA CAG GCA TTG CCT GAA AAG AGT ATA AAA GAA TTTCAG GAT TTT CCA TGG CGG ATG TGT GAC ATA C-3′. Primer 2 contained a Xho I restriction site (underlined) followed by 20 nt of the SFV sequence (in italics): SEQ ID NO. 21: 5′-CTC GAG GAT ATC CAA GAT GAG TGT GT-3′. A DNA fragment with 342 by was generated by PCR and cloned directly into the pGEM-T-easy plasmid to generate pGEM-Te-SFV-1. The absence of PCR errors in this plasmid was confirmed by sequencing. The 342 by fragment was released from pGEM-Te-SFV-1 by digestion with Spe I and Xho I and cloned into pGL3/AFP digested with the same enzymes to yield pGL3/AFP-SFV-1, which possesses the complete AFP promoter (217 bp) and enhancer (785 bp) followed by the 5′ end sequence of SFV (SFV-1, comprising 292 bp). An AFP-SFV-1 (1342 bp) cassette was obtained from pGL3/AFP-SFV-1 by Mlu I/Xho I digestion, treated with Klenow and cloned into pBS-SK+ digested with EcoR V, generating pBS/AFP-SFV-1. The SV40 late polyA (262 bp) was removed from pGL3/AFP by Xba I/BamH I digestion, blunted with Klenow and inserted into the Sal I site of pBS/AFP-SFV-1 also blunted with Klenow, thus giving rise to pBS/AFP-SFV-1-pA. A polylinker containing both unique Apa I and Nru I sites was inserted between Bam HI and Xma I sites in pBS/AFP-SFV-1-pA. The 3′ end sequence of SFV comprising 7985 bp was removed by digestion with Spe I/EcoR V from pBK-SFV-1, blunted with Klenow and inserted into the EcoR V position of pBS/AFP-SFV-1-pA, giving rise to pBS/AFP-SFV-pA.
LacZ reporter gene was obtained from pCMVb by digestion with Not I, treated with Klenow and inserted into the BamH I site of pBS/AFP-SFV-pA treated with Klenow to form pBS/AFP-SFV-LacZ-pA. A mIL-12 cassette containing the genes encoding for the p35 and p40 subunits linked by the internal ribosome entry site (IRES) was separated from pBS/mIL-12 by digestion with Spe I/Xho I, treated with Klenow and inserted into the BamHI position of pBS/AFP-SFV-mIL-12-pA, also blunted with Klenow, the plasmid pBS/AFP-SFV-mIL-12-pA being generated.
Construction of Hybrid Gutless Adenoviral Vectors
Four gutless adenoviral vectors have been constructed, as shown in FIG. 2. AFP-SFV-lacZ and AFP-SFV-mIL-12 contain a recombinant SFV replicon sequence controlled by the AFP promoter and enhancer. In these vectors the reporter gene LacZ or the therapeutic gene mIL-12 were cloned under the control of the SFV subgenomic promoter, respectively. AFP-lacZ and AFP-mIL-12 are also gutless adenovirus vectors, which contain LacZ and mIL-12 genes, directly controlled by the AFP promoter/enhancer, respectively. The process followed for the construction of these vectors is described below. In order to generate an adenoviral vector with sufficient cloning space to house the AFP-SFV-IL-12 expression cassette, the pSTK120 plasmid containing the sequence of a gutless adenovirus was modified. To that end, a 9-kb fragment was eliminated from the pSTK120 plasmid via digestion with Apa I. In addition, a polylinker containing the sites Asc I and Sbf I was inserted into this new plasmid, giving rise to pTGC3001. This plasmid contains the left ITR, the packaging signal, stuffer DNA from HPRT and C346, and the right ITR. The AFP-SFV-LacZ cassette was separated by digestion with Apa I from pBS/AFP-SFV-lacZ-pA and inserted in the Apa I site of pTGC3001, giving rise to pTGC3011. Similarly, the AFP-SFV-mIL-12 cassette was released from pBS/AFP-SFV-mIL-12-pA by digestion with BssH II, treated with Klenow and inserted in the Asc I site of pTGC3001, likewise treated with Klenow to generate pTGC3012.
Construction of Control Vectors
Construction of the Gutless Adenoviral Vector AFP-LacZ
The AFP enhancer/promoter (AFP p+e) sequence was separated from pGL3/AFP by digestion with Mlu I/Xho I, treated with Klenow and inserted into pCMVb, which had been previously digested with EcoRI/Xho I and treated with Klenow. In this way the CMV immediate early promoter was removed from pCMVb and substituted by AFP (p+e) to generate pAFPb. The AFP-LacZ cassette (5077 bp) was subsequently removed from pAFPb by digestion with Xba I/Nar I, treated with Klenow, and inserted into the Swa I site of pSTK120, also blunted with Klenow, giving rise to pTGC3013.
Construction of the Gutless Adenoviral Vector AFP-mIL-12
The mIL-12 cassette was removed from pBS/mIL-12 by digestion with Xho I/Spe I, and inserted in pGL3/AFP previously digested with Xho I/Xba I, which eliminated the luciferase gene from this latter plasmid and generated pAFP-mIL-12. The AFP-mIL-12 cassette (3760 bp) was removed from pAFP-mIL-12 by digestion with BamH I/Sca I, treated with Klenow and inserted in pSTK120 digested by Swa I, and likewise treated with Klenow to generate pTGC3014.
Rescue of the Gutless Adenoviral Vectors
Following Pme I digestion, phenol/chloroform extraction, and ethanol precipitation, 2 μg of pTGC3011, pTGC3012, pTGC3013, or pTGC3014 DNA were transfected into 293Cre4 cells, respectively. After transfection, cells were infected with helper virus AdLC8cluc. Subsequent large-scale amplification and preparation steps were performed as previously described (Philip Ng., Robin J. Parks, and Frank L. Graham. Preparation of helper-dependent adenoviral vectors. Methods in Molecular Medicine, Vol. 69, Gene Therapy Protocols, 2nd. Ed. 69, 371-88, 2002; H. Zhou, L. Pastore, A. L. Beaudet. Helper-dependent adenoviral vectors. Methods in Enzymology, vol, 346, 177-198, 2002; Hillgenberg M., et al. System for efficient helper-dependent minimal adenovirus constructions and rescue. Hum Gene Ther., 12; 643-657, 2001). All vector preparations were purified twice by CsC1 gradient centrifugation. The purified DNA vectors were analyzed by digestion with restriction enzymes and showed no sequence rearrangements. Titration of gutless adenovirus and helper virus contamination was evaluated using quantitative PCR. The proportion of total viral particles versus the infectious units (iu) was 20:1. Contamination by helper virus particles was approximately 0.5-1%.
Quantitative PCR
To determine the degree of helper virus contamination, a probe and primers for the quantitative PCR of the Ad5 E4 region were designed using the TaqMan program (TaqMan Probe #2), and were synthesized by Sigma-Genosys Ltd. (primer) and Applied Biosystems (probe). To determine the titer of gutless adenoviruses, probes and primers for the quantitative PCR of the LacZ and mIL-12 sequences from mice were designed using the TaqMan program (TaqMan Probe #2), and were synthesized by Sigma-Genosys Ltd (primer) and Applied Biosystems (probe). To determine the contamination by wild-type Ad, probes and primers for the quantitative PCR of the Ad5 E1 region were designed using the TaqMan program (TaqMan Probe #2), and were synthesized by Sigma-Genosys Ltd. (primer) and Applied Biosystems (probe).
In Vitro Experiments
Transgenic Expression in Cells Infected with Gutless Adenoviral Vectors
Cell lines derived from HCC (Hep3B, Huh7, HepG2, and PLC/PRF/5) and cell lines that are not derived from HCC (A549, HeLa, MHC1 and Clone 9) or cell lines that are derived from HCC but do not express AFP (SK-Hep-1) were infected with each of the four gutless adenoviral vectors (AFP-LacZ, AFP-SFV-LacZ, AFP-mIL-12, or AFP-SFV-mIL-12) at “moi” 1000, 100, or 10 (particles/cell), respectively. Three first generation adenoviruses (Ad/CMV-mIL-12, Ad/CMV-LacZ Ad/AFP-LacZ) were used as control. Supernatants from cells infected with mIL-12 vectors and lysates from cells infected with LacZ vectors were collected in duplicate from wells for determination of mIL-12 and β-galactosidase (β-gal) levels, respectively. Cells infected with LacZ vectors were also stained with X-gal. The mIL-12 level (p70) was measured with an ELISA kit (Pharmingen, San Diego, Calif.). The β-gal level was measured with an ELISA kit (Roche, Switzerland). The time course of mIL-12 expression was evaluated in HCC cells (Hep3B, Huh7) after infection with AFP-mIL-12, AFP-SFV-mIL-12, or with the control vector Ad/CMV-mIL-12 at a “moi” of 1000. Supernatants were collected daily until 5 days after the infection.
Analysis of the Specificity of Transgenic Expression Using Ad-SFV Hybrid Vectors In Vitro
To examine the specificity of transgenic expression with the previously described recombinant vectors, four human HCC cell lines (Hep3B, HepG2, Huh-7 and PLC/PRF/5) and two human cell lines that are not derived from HCC (HeLa and A549) or that are derived from HCC but do not express AFP (SK-Hep-1) were infected with AFP-mIL-12, AFP-SFV-mIL-12 or Ad-CMV-mIL-12 as positive control at different “moi” (10, 100, or 1000). Two days after infection, the supernatant was collected and its mIL-12 content was determined. The results are shown in FIG. 3 (A) and (B). No mIL-12 expression was observed in the human HCC cells when infected with AFP-mIL-12 at “moi” 10 or 100, and only at “moi” 1000 a very low level of mIL-12 was seen in some cell lines (FIG. 3A). In contrast, the infection of these cells with AFP-SFV-mIL-12 at “moi” 10, 100, or 1000 gave rise to the expression of mIL-12 in a dose-dependent manner (FIG. 3A). The level of mIL-12 expression in cells infected with “moi” 10 of AFP-SFV-mIL-12 was comparable to the level obtained in cells infected with AFP-mIL-12 at “moi” 1000. In addition, the level of mIL-12 in HCC cells infected with AFP-SFV-mIL-12 at different “moi” was comparable to that obtained with the control vector Ad-CMV-mIL-12. However, the infection of cells that do not express AFP with AFP-mIL-12 or AFP-SFV-mIL-12 yielded no detectable levels of mIL-12, even when using the highest “moi” (1000) (FIG. 3B). In these cells, only the control vector Ad-CMV-mII-12 was able to generate a high level expression of mIL-12.
On the other hand, four HCC cell lines (Hep3B, Huh-7, HepG2 and PLC/PRF/5) were infected with hybrid vectors of LacZ—AFP-lacZ, or AFP-SFV-lacZ—at different “moi” (10, 100, or 1000), and the specific expression of β-gal was determined. Similar data were likewise obtained in this case, the results of which are shown in FIG. 4.
FIG. 5 shows microphotographs of HCC cells infected with the gutless adenoviral vectors AFP-lacZ and AFP-SFV-lacZ, followed by staining with X-gal. The infection of HCC cells with AFP-lacZ resulted in a low level expression in the infected cells, which were only weakly stained. In contrast, the infection of HCC cells with AFP-SFV-lacZ led to a high level expression of β-gal, reflected by intense staining with X-gal. This data indicates that a hybrid vector Ad-SFV comprising an SFV replicon under the control of the AFP promoter may give rise a high level of intense transgenic expression in tumor cells that express AFP.
Time Course of the Expression of mIL-12 in HCC Cells In Vitro
To study the production of mIL-12 at different times following infection with the Ad-SFV hybrid vectors, two HCC cell lines (Hep3B and Huh-7) were infected with AFP-mIL-12 or with AFP-SFV-mIL-12, and the supernatants were collected daily for 5 days following infection. FIG. 6 shows the transgenic expression results obtained after the infection of the mentioned cells. Said results reflect a constant increase in the expression of mIL-12 from day 1 to day 4 after the infection in cells infected with AFP-SFV-mIL-12 (FIG. 6). However, on day 5 following infection, the mIL-12 levels decreased slightly. In the cells infected with AFP-mIL-12, the levels of expression were very low, and only a slight increase in the production of mIL-12 was noted over time.
Cytotoxicity Testing—Evaluation of Cell Proliferation by MTT Incorporation
HCC cells (Hep3B, Huh7, MCH-RH7777, Hep1-6) were infected with AFP-LacZ, AFP-SFV-LacZ, AFP-mIL-12, AFP-SFV-mIL-12, or Ad/CMV-mIL-12 at “moi” 1000. Five days after the infection, cell survival was determined by an MTT assay (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) Mosmann, T. (1983) J. Immunol. Meth. 65, 55-63; Tada, H. et al. (1986) J. Immunol. Meth. 93, 157-65. Briefly, cells were washed once with PBS and 200 μl of freshly prepared MTT dye solution was added per well (in 48-well plates). Cells were further cultured for 3-4 hours followed by addition of 500 μl of solubilization buffer. 100 μl of each sample were taken for the measurement of absorbance in a spectrophotometer at a wavelength of 570 nm.
Induction of Cell Death Following HCC Cell Infection with Ad-SFV Hybrid Vectors In Vitro
It has been described that the replication of SFV vectors induces cell death mediated by apoptosis in most cells of vertebrate origin. In order to evaluate whether this is also the case in HCC cells infected with Ad-SFV hybrid vectors, Hep3B and Huh-7 cells were infected with these vectors and cell survival was determined on day 5 following infection. As can be seen in FIG. 7, survival at this post-infection timepoint was less than 20% in cells infected with AFP-SFV-mIL-12, or with AFP-SFV-lacZ. However, infection of these same cells with AFP-mIL-12 or AFP-lacZ, or with the control vector AdCMVmIL-12, did not affect cell survival. These results indicate that SFV replicates in cells infected with AFP-SFV vectors.
Detection of SFV Rep by Immunofluorescence
HCC cells (Hep3B, Huh7, MCH-RH7777) were seeded on glass coverslips in 6-well plates (1×105 cells/well) and infected with AFP-mIL-12, AFP-SFV-mIL-12, or Ad/CMV-mIL-12 at “moi” 1000. Two days after infection coverslips were washed twice with PBS, and cells were fixed with methanol at −20° C. for 6 min. The plates were again washed three times with PBS and incubated at room temperature (RT) during 30 min. with PBS containing 0.5% gelatin and 0.25% BSA to block nonspecific binding. The blocking buffer was then replaced with the primary antibody (anti-replicase MAb) diluted 1:10 in blocking buffer, and was incubated at RT for 30 min. The cells were again washed three times with PBS-0.25% BSA, and incubated for 30 min. at RT with the secondary antibody (antimouse rabbit serum conjugated with FITC, Sigma) diluted 1:250 in blocking buffer. Finally, the cells were washed three times with PBS-0.25% BSA, once with water, and arranged on glass slides using Vecta shield with Dapi in order to stain the cell nuclei.
Expression of SFV Rep in HCC Cells Infected with Ad-SFV Hybrid Vectors In Vitro
The expression of SFV Rep was examined in HCC cells infected with Ad-SFV hybrid vectors via immunofluorescence with a specific monoclonal antibody for this protein. FIG. 8 shows that the HCC cells infected with AFP-SFV-mIL-12 or AFP-SFV-lacZ exhibited intense cytoplasmic positivity for Rep. In contrast, the cells infected with AFP-mIL-12 or AFP-lacZ showed no staining.
In Vivo Experiments
Induction of HCC Xenografts and Study of In Vivo Gene Transfer Efficiency and Specificity
Huh-7 cells were collected and washed twice with serum-free medium. 2×106 cells were resuspended in 100 μl saline serum and injected subcutaneously (s.c.) into the right flank of BALB/c nude mice. Four weeks after the inoculation of the cells, and when tumor nodules reached 6-8 mm in diameter, 1×1010 viral particles of AFP-LacZ (n=4) or of AFP-SFV-LacZ (n=4) diluted in 80 μl of saline were injected intratumorally. Control animals (n=3) were injected intratumorally with 80 μl of saline. Mice were sacrificed on day 3 or 6 after the inoculation. At these times the tumors and livers were taken from each animal, embedded in O.C.T. (Sakura, Holland), and frozen at −80° C. Frozen tissues were sectioned and placed on glass slides to be stained with X-gal or analyzed by TUNEL., To study the specificity of the infection with the hybrid vector in vivo, normal Balb/c mice were injected intravenously with AFP-LacZ (n=4), AFP-SFV-LacZ (n=4) or Ad/CMV-LacZ at a dose of 1010 viral particles/mouse. The animals were sacrificed on the third day after the inoculation and the main organs were collected to analyze the expression of LacZ by means of staining with X-gal.
Efficiency of Gene Transfer of Ad-SFV Hybrid Vectors in Human HCC Xenografts in Nude Immunodeficient Mice
To study the efficiency of the transduction of gutless hybrid vectors in vivo, a human HCC model based on Huh7 cells capable of expressing AFP was employed. The Huh7 cells were inoculated subcutaneously in nude immunodeficient Balb/c mice, and after the generation of tumor nodules after 30 days, the animals were injected intratumorally with 1×1010 viral particles of AFP-SFV-LacZ or AFP-LacZ as control. The mice were sacrificed 3 or 6 days after the injection of the virus, and both the tumor and liver were removed and examined by staining with X-gal. As can be seen in FIG. 9A, there is a weak transgenic expression in tumor sections of animals that have received AFP-lacZ. In contrast, there is an intense expression of LacZ in tumor sections of animals that received AFP-SFV-LacZ (FIG. 9B). No transgenic expression was observed in liver sections from animals that received either AFP-LacZ or AFP-SFV-LacZ, indicating that the vectors were probably confined in the inoculation site (data not shown). With the aim of studying whether the Ad-SFV hybrid vectors induce apoptosis in infected tumor cells, treated tumor sections were analyzed by means of the TUNEL technique. No apoptosis was observed in the samples from mice inoculated with AFP-LacZ (FIG. 9C). However, an abundant amount of apoptotic cells was observed in the tumors of animals who had received AFP-SFV-LacZ (FIG. 9D). This data indicates that Ad-SFV hybrid vectors not only induce gene expression specifically in tumors but they also induce selective cell death by apoptosis in these same cells.
In Vivo Specificity of Ad-SFV Hybrid Vectors
To show the specificity of the hybrid vectors, 1010 viral particles of AFP-LacZ, AFP-SFV-LacZ or of the control vector Ad/CMV-LacZ were administered intravenously in Balb/c mice. Three days after the administration of the vectors, the expression of β-galactosidase in liver was analyzed. As shown in FIG. 9 (E-F), neither AFP-LacZ nor AFP-SFV-LacZ were able to induce detectable expression of the transgene in liver. However, a high proportion of β-galactosidase positive cells in hepatic tissue sections was observed in those animals that received Ad/CMV-LacZ (FIG. 9G). These data confirm that the expression mediated by the hybrid vectors is specific for tumor cells.
Orthotopic HCC Induction and Gene Therapy In Vivo
5×105 McA-RH7777 cells were inoculated in the left lobe of the liver of Buffalo rats. Ten days after the inoculation of the tumor cells, the appearance of a single tumor nodule of 7-10 mm in diameter was observed in each animal. The tumors were treated with 1011 or 2×1011 viral particles of AFP-mIL-12, AFP-SFV-mIL-12, or with saline solution as control. Two and four weeks after treatment, the animals were anesthetized and subjected to laparotomy to observe the evolution of the tumor. An analysis of animal survival was also made. The size of the tumors was assessed by measuring the length and width of each nodule and applying the formula: Tumor volume=(length in mm)×(width in mm)2×0.5236 (Janik et al., 1975).
Efficiency of Orthotopic HCC Treatment in Buffalo Rats
In order to investigate the anti-tumor efficiency of the Ad-SFV hybrid vector carrying IL-12, orthotopic HCC tumors were established in rats by implanting rat McH-RH7777 cells in the liver. This model was chosen because it has been demonstrated that McH-RH7777 cells express AFP. In a first experiment, the animals were treated with a single intratumor injection of 1×1011 viral particles of AFP-mIL-12, AFP-SFV-mIL-12, or with saline solution as control (FIG. 10A-C). The animals that received AFP-mIL-12 showed a reduced tumor size compared with the control animals, which suffered a constant increase in tumor size throughout the experiment (FIG. 10A-B). However, treatment with AFP-SFV-mIL-12 resulted in a complete regression of the tumor in 1 of 4 treated rats, stabilization of the disease in two, and an absence of response in 1 animal (FIG. 10C). For the purpose of verifying whether larger doses of the hybrid vector could increase the anti-tumor effect, a second experiment was carried out in which the animals were treated intratumorally with a dose of 2×1011 viral particles of AFP-mIL-12, AFP-SFV-mIL-12, or with saline solution as control (FIG. 10 D-G). As in the previous experiment, the animals that received the AFP/IL-12 vector showed only a slight anti-tumor response, which translated into only one complete remission, 4 animals with tumors that grew more slowly than in the controls, and 7 animals with no response, of a total of 12 treated animals (FIG. 10E). However, treatment with the AFP-SFV-IL-12 vector had a much more potent effect, inducing a complete tumor regression in four animals (33%), partial regression in 6 animals (50%), delayed tumor growth progression in 2 animals (16%), and no response in another 2 animals (16%), of a total of 12 treated animals (FIG. 10F). In this second study the AFP/SFV-IL-12 vector allowed the survival of 50% of the animals treated, versus 0% survival among the animals treated with AFP-IL-12 or with saline solution (FIG. 10G).
In Vivo Toxicity Study: Determination of Serum Transaminases and IL-12 Levels, and Liver Histological Evaluation
Blood samples were collected from the rats treated intratumorally with the adenoviral vectors AFP-SFV-IL-12 or AFP-IL-12 at a dose of 2×1011, or with saline solution, 4 and 8 days after the inoculation. This study also included rats inoculated with 108 particles of the alphavirus SFV-IL-12. Serum was separated from blood by centrifugation at 2000 rpm for 15 minutes. Transaminase levels were determined using a Hitachi 911 Automatic Analyzer (Boehringer Mannheim, Germany). The IL-12 levels were determined by ELISA. The histological study was carried out by the extraction of the liver in the animals inoculated three days after the treatment. The organ was fixed in formol, embedded in paraffin and sectioned to a thickness of 6 microns with a microtome. These sections were then stained with hematoxylin/eosin.
Study of the Toxicity of Ad-SFV Hybrid Vectors in Rats
To evaluate toxicity associated with the administration of the AFP-SFV-IL-12 hybrid vector, the levels of transaminases (GOT, GPT and GGTL) were determined in the serum of rats treated intratumorally with 2×1011 viral particles of the different vectors (see previous section). This study also included a group of rats also inoculated intratumorally with 108 viral particles of alphaviral vector SFV-IL-12 (FIG. 11A). The rats inoculated with the AFP-SFV-IL-12 or AFP-IL-12 adenoviral vectors showed very low transaminase levels that were very similar to the levels of control animals inoculated with saline solution. However, the transaminase levels in the animals treated with the particles of SFV-IL-12 were significantly greater than in the other groups (p<0.05). In this study, determinations of the IL-12 level present in the serum of the animals at the same timepoints were also made. No IL-12 was detected in the serum of the animals inoculated with the Ad AFP-SFV-IL-12 or AFP-IL-12 vectors, or with saline solution (FIG. 11B), indicating that expression of the transgene in these vectors is restricted to the tumors, and suggesting that the toxicity of the Ad-SFV hybrid vector is very low. Treatment with the SFV-IL-12 viral particles, however, induced high serum levels of IL-12 after short periods of time, a situation that could cause liver toxicity. Finally, the toxicity study was completed with a histological analysis of liver sections stained with hematoxylin/eosin from rats treated intratumorally with the same vectors and at the same doses already described (FIG. 12). This study showed no histological differences between the rats that received saline solution and those that received the AFP-SFV-IL-12 or AFP-IL-12 adenoviral vectors. However, areas with eosinophilic hepatocytes as well as the fusion of these hepatocytes was observed in liver sections of rats treated with SFV-IL-12 viral particles, indicating a certain degree of toxicity (black arrows, FIG. 12D).

## Claims

1. A genic expression adenoviral hybrid vector for expressing a gene of interest directly in an adenovirus-permissive cell in which a tissue-specific promoter is active, wherein said tissue specific promoter is the tumor specific promoter AFP, characterized in that the genic expression adenoviral hybrid vector comprises at least the following elements, oriented in the direction 5′ to 3′:
i. a first chain of adenoviral origin comprising a first inverted terminal repeat (ITR) sequence and a signal sequence for packaging of the adenovirus;
ii. a first non-encoding stuffer sequence;
iii. a sequence corresponding to a tissue specific promoter, wherein said tissue specific promoter is the tumor specific promoter AFP;
iv. a chain of cDNA derived from an alphavirus, the sequence of which is partly complementary to an alphaviral RNA sequence, comprising at least a sequence encoding for at least one exogenous gene of interest; said chain of cDNA derived from an alphavirus comprising:
(a) a 5′ sequence necessary for replication of the alphavirus,
(b) a sequence encoding for the non-structural proteins required for replication of the alphaviral RNA,
(c) at least one subgenomic promoter of the alphavirus, and
(d) a 3′ sequence necessary for replication of the alphavirus;
wherein said sequence encoding for at least one exogenous gene of interest is under the control of said at least one subgenomic promoter of the alphavirus; and
said chain of cDNA derived from an alphavirus is functionally controlled by the promoter iii;


v. a polyadenylation sequence; and
vi. a second adenoviral inverted terminal repeat (ITR) sequence.

2. A genic expression adenoviral hybrid vector according to claim 1, further comprising an element vii which is a second non-encoding stuffer sequence located between element v and element vi.

3. A genic expression adenoviral hybrid vector according to claim 1, characterized in that element ii is a human non-encoding stuffer sequence.

4. A genic expression adenoviral hybrid vector according to claim 3, characterized in that element ii is the intron region of human genomic hypoxanthine phosphoribosyltransferase, HPRT.

5. A genic expression adenoviral hybrid vector according to claim 1, characterized in that element i comprises the sequence SEQ ID NO: 1.

6. A genic expression adenoviral hybrid vector according to claim 1, characterized in that element iii is a tumor specific promoter having the sequence SEQ ID NO: 7, corresponding to AFP p+e.

7. A genic expression adenoviral hybrid vector characterized in that the genic expression adenoviral hybrid vector comprises at least the following elements, oriented in the direction 5′ to 3′:
i. a first chain of adenoviral origin comprising a first inverted terminal repeat (ITR) sequence and a signal sequence for packaging of the adenovirus;
ii. a first non-encoding stuffer sequence;
iii. a sequence corresponding to a tissue specific promoter, wherein said tissue specific promoter is the tumor specific promoter AFP;
iv. a chain of cDNA derived from an alphavirus, the sequence of which is partly complementary to an alphaviral RNA sequence, comprising at least a sequence encoding for at least one exogenous gene of interest; said chain of cDNA derived from an alphavirus comprising:
(a) a 5′ sequence necessary for replication of the alphavirus,
(b) a sequence encoding for the non-structural proteins required for replication of the alphaviral RNA,
(c) at least one subgenomic promoter of the alphavirus, and
(d) a 3′ sequence necessary for replication of the alphavirus;
wherein said sequence encoding for at least one exogenous gene of interest is under the control of said at least one subgenomic promoter of the alphavirus; and
said chain of cDNA derived from an alphavirus is functionally controlled by the promoter iii;


v. a polyadenylation sequence; and
vi. a second adenoviral inverted terminal repeat (ITR) sequence;
wherein said at least one exogenous gene of interest is expressed directly in an adenovirus-permissive cell in which a tissue-specific promoter is active, and wherein said tissue specific promoter is the tumor specific promoter AFP.

8. The genic expression adenoviral hybrid vector according to claim 1, wherein said element iv comprises a sequence derived from the Semliki Forest Virus (SFV).

9. A genic expression adenoviral hybrid vector according to claim 8, characterized in that element iv a), b) and c) comprises, as a whole, a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.

10. A genic expression adenoviral hybrid vector according to claim 9, characterized in that element iv d) has the sequence SEQ ID NO: 5.

11. A genic expression adenoviral hybrid vector according to claim 1, characterized in that the exogenous gene of interest is selected from the group consisting of one or more therapeutic genes, one or more reporter genes, and combinations thereof.

12. A genic expression adenoviral hybrid vector according to claim 11, characterized in that the exogenous gene of interest is the mammalian interleukin IL-12 therapeutic gene.

13. A genic expression adenoviral hybrid vector according to claim 11, characterized in that the exogenous gene of interest is the human interleukin hIL-12 therapeutic gene.

14. A genic expression adenoviral hybrid vector according to claim 9, characterized in that element iv comprises (subgenomic promoter+exogenous gene of interest) in series or in several subsets.

15. A genic expression adenoviral hybrid vector according to claim 1, characterized in that element iv forms a replicon functionally controlled by the promoter iii, and in that the alphaviral subgenomic promoter in iv.c) functionally controls the expression of the exogenous gene of interest.

16. A genic expression adenoviral hybrid vector according to claim 1, characterized in that element v is a polyadenylation sequence of SV40.

17. A genic expression adenoviral hybrid vector according to claim 16, characterized in that element v comprises the sequence SEQ ID NO: 6.

18. A genic expression adenoviral hybrid vector according to claim 2, characterized in that the second non-encoding stuffer sequence is C346.

19. A genic expression adenoviral hybrid vector comprising:
i. a first chain of adenoviral origin comprising a first inverted terminal repeat (ITR) sequence and a signal sequence for packaging of the adenovirus;
ii. a first non-encoding stuffer sequence, which is the intron region of human genomic hypoxanthine phosphoribosyltransferase (HPRT);
iii. a sequence corresponding to a tissue specific promoter, which is the AFP promoter,
iv. a cDNA chain derived from an alphavirus, the sequence of which is partly complementary to an alphaviral RNA derived from the SFV virus, which comprises a sequence encoding for an exogenous gene of interest which is hIL-12,
v. a polyadenylation sequence of SV40,
vi. a second adenoviral inverted terminal repeat (ITR) sequence and
vii. a second non-encoding stuffer sequence, which is human genomic C346, located between element v and element vi.

20. A genic expression adenoviral hybrid vector according to claim 1, characterized in that said vector has a length comprised between 27 and 38 kilobases.

21. A genic expression adenoviral hybrid vector characterized in that the genic expression adenoviral hybrid vector comprises at least the following elements, oriented in the direction 5′ to 3′:
i. a first chain of adenoviral origin comprising a first inverted terminal repeat (ITR) sequence and a signal sequence for packaging of the adenovirus;
ii. a first non-encoding stuffer sequence;
iii. a sequence corresponding to a tissue specific promoter;
iv. a cDNA chain derived from an alphavirus, the sequence of which is partly complementary to an alphaviral RNA sequence derived from the SFV virus, comprising at least a sequence encoding for at least one exogenous gene of interest;
v. a polyadenylation sequence;
vi. a second adenoviral inverted terminal repeat (ITR) sequence; AND
vii. a second non-encoding stuffer sequence located between element v and element vi,

wherein the vector comprises the sequence of SEQ ID NO:8.

22. A method of transferring genetic material to a cell comprising the steps of administering the adenoviral hybrid vector of claim 1 to a subject.

23. The method according to claim 22 wherein said cell is a tumor cell.

24. The method according to claim 23 wherein said cell is a tumor cell expressing AFP.

25. A method of treating hepatocarcinoma tumors in a subject comprising the step of directly administering the adenoviral vector of claim 1 comprising a nucleotide sequence encoding IL-12 to said tumor in a subject.

26. A method for inducing an immune response against foreign antigens comprising the steps of administering said adenoviral hybrid vector of claim 1 to a subject.

27. A pharmaceutical composition comprising at least one adenoviral hybrid vector according to claim 1.

28. The pharmaceutical composition according to claim 27, characterized in that said composition comprises at least one adenoviral hybrid vector defined according to claim 1, wherein the exogenous gene of interest is hIL-12.

29. A method of inducing an immune response against foreign antigens in a subject comprising the steps of administering to said subject the pharmaceutical composition of claim 27, the hybrid vector further comprising a sequence encoding a foreign antigen of interest.