Intratumor RNA interference of cell cycle genes slows down tumor progression.

Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing.

Gene therapy of Hunter syndrome: evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS).

Mucopolysaccharidosis type II (MPSII) is an inherited disorder due to a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The disease is characterized by a considerable deposition of heparan- and dermatan-sulfate, causing a general impairment of physiological functions. Most of the therapeutic protocols proposed so far are mainly based upon enzyme replacement therapy which is very expensive. There is a requirement for an alternative approach, and to this aim, we evaluated the feasibility of muscle electro gene transfer (EGT) performed in the IDS-knockout (IDS-ko) mouse model. EGT is a highly efficient method of delivering exogenous molecules into different tissues. More recently, pre-treatment with bovine hyaluronidase has shown to further improve transfection efficiency of muscle EGT. We here show that, by applying such procedure, IDS was very efficiently produced inside the muscle. However, no induced IDS activity was measured in the IDS-ko mice plasma, in contrast to matched healthy controls. In the same samples, an anticipated and rapidly increasing immune response against the recombinant protein was observed in the IDS-ko vs control mice, although reaching the same levels at 5 weeks post-injection. Additional experiments performed on healthy mice showed a significant contribution of hyaluronidase pre-treatment in increasing the immune response.

CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge.

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.

In vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors.

Baculovirus vectors are efficient tools for gene transfer into mammalian cells in vitro. However, in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system. To characterize further the gene transfer efficacy of baculovirus we examined the vector transduction efficiency in skeletal muscle. Vectors expressing vesicular stomatitis virus glycoprotein (VSV-G) in the viral envelope were generated by inserting the VSV-G coding sequence downstream of the polyhedrin promoter. Two viruses were constructed to carry either the Escherichia coli beta-galactosidase (beta-Gal) gene or the mouse erythropoietin (EPO) cDNA cloned downstream of the cytomegalovirus immediate-early promoter and enhancer. The greater gene transduction efficiency of the Bac-G-betaGal vector was confirmed by comparing the beta-Gal expression level in a variety of human and mouse cell lines with that obtained on infection with Bac-betaGal, a vector that lacks VSV-G. Similarly, a 5- to 10-fold increase in beta-Gal expression between Bac-G-betaGal and Bac-betaGal was observed when mouse myoblasts and myotubes were infected. The same increase in beta-Gal expression was detected on injection of the Bac-G-betaGal vector in the quadriceps of BALB/c and C57BL/6 mice. In contrast, a 2-fold difference in transduction was observed between these two vectors in DBA/2J mouse strain. Last, expression of EPO cDNA was detected for at least 178 days in DBA/2J mice on Bac-G-EPO injection into the quadriceps whereas EPO expression declined to normal values by 35 days postinfection in BALB/c and C57BL/6 mice. Thus, these results indicate that baculovirus may be considered a useful vector for gene transfer in mouse skeletal muscle and that persistence of expression may depend on the mouse strain used.

Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector.

Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose-response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.

Gene electrotransfer results in a high-level transduction of rat skeletal muscle and corrects anemia of renal failure.

We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroinjection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secretion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be applied to produce therapeutically significant levels of erythropoietin in chronic renal failure.

A human antibody specific for hepatitis C virus core protein: synthesis in a bacterial system and characterization.

The cDNA coding for the Fab fragment of the human B12.F8 antibody (Ab), directed against the putative nucleocapsid component (core protein) of hepatitis C virus (HCV), was cloned in the prokaryotic phagemid vector, pHEN-1, to obtain its expression in Escherichia coli. The functionality and specificity of the recombinant Ab, called B12Fab, were examined by Western blot and ELISA using recombinant HCV core protein as antigen. The specificity of B12Fab was further confirmed by ELISA with the 33-mer peptide epitope recognized by the original whole B12.F8 Ab. By immunofluorescence, the recombinant B12Fab was shown to recognize HCV core protein produced in cells transfected with HCV cDNA, indicating that the recombinant B12Fab is suitable as a diagnostic tool for tissue localization of the virus. The B12Fab also functioned when displayed on phage particles, providing the basis for future experiments of in vitro affinity maturation and selection of mutants. The variable chain coding regions of the recombinant B12Fab clone were sequenced and the V-gene usage was determined by comparison with the V kappa and VH germline sequences. The B12Fab V kappa chain belongs to the subgroup II and shows the highest degree of homology with the A3 germline gene, whereas the sequence of the VH chain is strictly related to that of the Humhv3019b18 gene of the VH3 family. These results are, to our knowledge, the first report of molecular cloning and characterization of a functional human Ab specific for an HCV antigen.

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

Towards the use of baculovirus as a gene therapy vector.

The use of baculovirus vectors for gene expression in mammalian cells is in continuous expansion. These vectors do not replicate in mammalian cells, do not cause a cytopathic effect upon infection and are able to carry large DNA inserts. Baculovirus vectors have been shown to transduce various cell types in vitro and in vivo with significant efficiency leading to stable gene expression. This review focuses on recent developments with baculovirus that highlight its potential usefor new gene therapy strategies.

Murine coronavirus gene 1 polyprotein contains an autoproteolytic activity.

The 5' most gene of the murine coronavirus genome, gene 1, is presumed to encode the viral RNA-dependent RNA polymerase. cDNA clones representing this gene encompass more than 22 kilobases, suggesting that this region may encode multifunctional polyprotein(s). It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To identify possible functional domains of gene 1 and further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of the murine coronavirus mouse hepatitis virus strain JHM were subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. Although p28 is encoded from the first 1 kilobase at the 5'-end of the genome, translation of in vitro transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3 kilobase region was synthesized. This result suggests that the region close to 5.3 kilobases from the 5'-end of the genomic RNA is essential for the proteolytic cleavage and may contain an autoproteolytic activity. Addition of the protease inhibitor ZnCl2 blocked cleavage of the p28 protein. Site-directed mutagenesis of Cys residue 1137 significantly reduced the cleavage of the p28 protein, indicating that this residue, probably in conjunction with a downstream domain, plays an essential role in the cleavage of p28. This Cys residue may be part of a papain-like autoprotease encoded by gene 1.

Non-viral transfer approaches for the gene therapy of mucopolysaccharidosis type II (Hunter syndrome).

AIMS: Hunter syndrome is a rare X-linked lysosomal storage disorder caused by the deficiency of the housekeeping enzyme iduronate-2-sulphatase (IDS). Deficiency of IDS causes accumulation of undegraded dermatan and heparan-sulphate in various tissues and organs. Approaches have been proposed for the symptomatic therapy of the disease, including bone marrow transplantation and, very recently, enzyme replacement. To date, gene therapy strategies have considered mainly retroviral and adenoviral transduction of the correct cDNA. In this paper, two non-viral somatic gene therapy approaches are proposed: encapsulated heterologous cells and muscle electro-gene transfer (EGT). METHODS: Hunter primary fibroblasts were co-cultured with either cell clones over-expressing the lacking enzyme or with the same incorporated in alginate microcapsules. For EGT, plasmid vector was injected into mouse quadriceps muscle, which was then immediately electro-stimulated. RESULTS: Co-culturing Hunter primary fibroblasts with cells over-expressing IDS resulted in a three- to fourfold increase in fibroblast enzyme activity with respect to control cells. Fibroblast IDS activity was also increased after co-culture with encapsulated cells. EGT was able to transduce genes in mouse muscle, resulting in at least a tenfold increase in IDS activity 1-5 weeks after treatment. CONCLUSION: Although preliminary, results from encapsulated heterologous cell clones and muscle EGT encourage further evaluations for possible application to gene therapy for Hunter syndrome.

An oral TLR7 agonist is a potent adjuvant of DNA vaccination in transgenic mouse tumor models.

In vivo electroporation of plasmid DNA (DNA-EP) is an efficient and safe method for vaccines resulting in increased DNA uptake, enhanced protein expression and increased immune responses to the target antigen in a variety of species. To further enhance the efficacy of DNA-EP, we have evaluated the toll-like receptor7 (TLR7) agonist-2, 9, substituted 8-hydroxyadenosine derivative or SM360320--as an adjuvant to vaccines against HER2/neu and CEA in BALB-neuT and CEA transgenic mice (CEA.Tg), respectively. SM360320 induced in vivo secretion of interferon alpha (IFNalpha) and exerted a significant antitumor effect in CEA.Tg mice challenged with a syngenic tumor cell line expressing CEA and an additive effect with a CEA vaccine. Additionally, combination of SM360320 with plasmid encoding the extracellular and transmembrane domain of ratHER2/neu affected the spontaneous tumor progression in BALB-neuT mice treated in an advanced disease setting. The antitumor effect in mice treated with DNA-EP and SM360320 was associated with an anti-CEA and anti-p185(neu) antibody isotype switch from IgG1 to IgG2a. These data demonstrate that SM360320 exerts significant antitumor effects and can act in association with DNA-EP for CEA-positive colon cancer and HER2-positive mammary carcinoma. These observations therefore emphasize the potential of SM360320 as immunological adjuvant for therapeutic DNA vaccines.

CD8+ T-cell tolerance can be broken by an adenoviral vaccine while CD4+ T-cell tolerance is broken by additional co-administration of a Toll-like receptor ligand.

T-cell tolerance to tumor antigens is a considerable challenge to cancer immunotherapy. The existence of a murine model transgenic for human carcinoembryonic antigen (CEA) allows CEA vaccination efficacy to be studied in a physiologically tolerant context. Immunization of CEA-transgenic mice with an adenoviral vector coding for CEA induced a significant CD8+ T-cell response specific to CEA but failed to induce CEA-specific CD4+ T cells and antibodies. To overcome CD4+ T-cell tolerance, we explored the effect of adjuvants inducing in vivo dendritic cell maturation. Two different Toll-like receptor ligands, monophosphoryl lipid A (MPL) and CpG motif-containing oligodeoxynucleotides (CpG-ODN), were tested. CD4+-mediated IFN-gamma production was induced in the CEA-transgenic mice only when the genetic immunization was performed in the presence of these adjuvants. Moreover, CpG-ODN had a greater effect than MPL in inducing CD4+ T-cell response and enabling anti-CEA antibody production.

Adenovirus transduction and culture conditions affect the immunogenicity of murine dendritic cells.

Adenovirus vectors encoding carcinoembryonic antigen (Ad-CEA) or costimulatory molecules CD80, intercellular adhesion molecule-1 (ICAM-1) and leucocyte function-associated antigen-3 (LFA-3) (Ad-STIM) were used to transduce murine bone marrow-derived dendritic cells (BMDC). BMDC were characterized for expression of activation markers and for their ability to elicit protective immunity against MC38-CEA tumours in wildtype and CEA-transgenic (CEA-tg) mice. To determine optimal culture conditions, studies were conducted using BMDC cultured in heterologous bovine serum or autologous mouse serum. Transduction of cells grown in presence of heterologous serum increased the expression of costimulatory molecules, major histocompatibility complex class II, of IL-6 and IL-12. Upon vaccination, tumour protection was not specific and was observed also with untransduced cells. Transduced BMDC cultured in the presence of autologous serum showed low expression of the activation markers, did not express IL-6 and had reduced ability to stimulate T-cell proliferation. Nonetheless, CEA-specific CD8+ T-cell response was enhanced upon coinfection of Ad-STIM and Ad-CEA in both mouse strains, although this immune response was not sufficient to protect CEA-tg mice from tumour challenge. These studies support the use of BMDC transduced with Ad vectors encoding tumour antigens for cancer immunotherapy and demonstrate that culture conditions greatly affect the immunological properties of these cells.

Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y.

A base change from C to U at position 472 of the 5' noncoding region of the poliovirus genome is known to be a major determinant of attenuation in the P3/Sabin vaccine strain. To determine the biochemical basis for the attenuated phenotype imparted by this mutation, a cell line in which replication of neurovirulent and attenuated viruses could be distinguished was identified. A pair of P3/Sabin-P2/Lansing viral recombinants that differ only at position 472 was used; the viruses replicated equally well in HeLa cells, but the virus with a U at base 472 was attenuated in mice. In the human neuroblastoma cell line SH-SY5Y, recombinants with a U at base 472 replicated to approximately 10-fold-lower titers than did neurovirulent viruses with a C at this position. Analysis of viral RNA and protein synthesis indicated that translation of the attenuated viral RNA was specifically reduced in SH-SY5Y cells.

Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.

Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.

Reduced mouse neurovirulence of poliovirus type 2 Lansing antigenic variants selected with monoclonal antibodies.

The Lansing strain of poliovirus type 2 is a mouse-adapted virus that induces a fatal paralytic disease in mice after intracerebral inoculation. Our previous results indicated that the mouse-adapted phenotype maps to the Lansing viral capsid. To further define regions of the capsid that are specifically involved in the infection of mice, antigenic variants resistant to neutralization with monoclonal antibodies were selected, and their mouse neurovirulence was studied. The monoclonal antibodies used were directed against antigenic site 1, an immunodominant loop of capsid polypeptide VP1 located on the virion surface. Ten of twenty-two variants selected had lower intracerebral neurovirulence in mice when compared to the parental virus. Four of the ten antigenic variants with reduced neurovirulence were temperature sensitive (ts) for replication in HeLa cells, while the remaining six variants replicated in HeLa cells as well as the parent virus. Two ts+ variants that were studied had a reduced ability to replicate in the mouse brain. There was no difference in the histopathology and pattern of involvement in the central nervous system of one variant compared to the parent virus. In three variants, reduction of neurovirulence correlated with specific amino acid substitutions at positions 100 and 101 of VP1, located within antigenic site 1. The ts phenotype in three variants was associated with a single amino acid deletion at position 105. Virus recovered from the brain of paralyzed mice that had been inoculated with the antigenic variants was characterized to identify the virus causing disease. In most cases, brain isolates resembled the inoculated virus in neurovirulence and amino acid sequence at the antigenic site. Virus recovered from brains of paralyzed mice that had been inoculated with the ts variants was either ts+ or cold sensitive, and had become more neurovirulent. These results suggest that specific amino acid changes within an antigenic site on the virion surface may result in reduction of mouse neurovirulence without affecting viral replication in cultured cells.

Coronavirus mRNA synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences.

The mRNA synthesis of mouse hepatitis virus (MHV) has been proposed to be the result of interaction between the leader RNA and the intergenic sites. Previously, we have identified a transcription initiation site (for mRNA 2-1), which is more efficiently transcribed by viruses containing two copies of UCUAA sequence in the leader RNA than by those with three copies. In this study, we have identified several sites which are regulated in the opposite way, namely, they are efficiently transcribed by the leader RNA with three UCUAA copies but not by those with two copies. These sites were characterized by primer extension and amplification by polymerase chain reaction. One of these sites is in the gene 3 region of a recombinant virus between A59 and JHM strains of MHV. Another is in the gene 2 region of MHV-1 strain. Both of these sites have a sequence similar to but different from the consensus transcription initiation signal (UCUAAUCUAUC and UUUAAUCUU, as opposed to UCUAAAC). These two novel intergenic sequences are not present in the genome of the JHM strain, consistent with the absence of these mRNAs in the JHM-infected cells. The discovery of this type of transcription initiation site provides additional evidence for the importance of the leader RNA in the transcription initiation of MHV mRNAs.

A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans.

A mutation in the genome of poliovirus type 3 that is known to reduce neurovirulence in humans similarly reduces neurovirulence in mice when incorporated into a mouse-adapted-human poliovirus recombinant. Viral recombinants with a uracil at nucleotide position 472 in the 5'-noncoding regions of their genomes are unable to replicate in the mouse brain. Viral recombinants with a cytosine at this position are neurovirulent in mice. Neurovirulence of poliovirus in mice may therefore prove to be a useful indicator of the genetic stability of new attenuating mutations created by site-directed mutagenesis.