Juraj Hlavaty

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105+ endothelial cells, human CD133+ hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133+ cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.

Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlytic retroviral replicating vector

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma (www.clinicaltrials.gov NCT01156584).

Toca 511 gene transfer and 5-fluorocytosine in combination with temozolomide demonstrates synergistic therapeutic efficacy in a temozolomide-sensitive glioblastoma model

Toca 511 (vocimagene amiretrorepvec), an amphotropic retroviral replicating vector (RRV), can successfully and safely deliver a functional, optimized cytosine deaminase (CD) gene to tumors in orthotopic glioma models. This agent, in conjunction with subsequent oral extended-release 5-fluorocytosine (5-FC) (Toca FC), is currently under investigation in patients with recurrent high-grade glioma . Temozolomide (TMZ) with radiation is the most frequently used first-line treatment for patients with glioblastoma, the most common and aggressive form of primary brain cancer in adults. However, subsets of patients with certain genetic alterations do not respond well to TMZ treatment and the overall median survival for patients who respond remains modest, suggesting that combinatorial approaches may be necessary to significantly improve outcomes. We show that in vitro TMZ delays but does not prevent RRV spread, nor interfere with Toca 511+5-FC-mediated cell killing in glioma tumor cells, and in vivo there is no significant hematologic effect from the combination of 5-FC and the clinically relevant dose of TMZ. A synergistic long-term survival advantage is observed in mice bearing an orthotopic TMZ-sensitive glioma after Toca 511 administration followed by coadministration of TMZ and 5-FC. These results provide support for the investigation of this novel combination treatment strategy in patients with newly diagnosed malignant glioma.

Multiple Modifications Allow High-Titer Production of Retroviral Vectors Carrying Heterologous Regulatory Elements

ABSTRACT Tumor-specific expression of therapeutic genes is a prerequisite in many approaches to retrovirus-mediated cancer gene therapy. However, tissue specificity is often associated with a reduction in viral titer. To overcome this problem, we constructed a series of murine leukemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors carrying either the simian virus 40 poly(A) signal trimer (3pA) inserted in the 3′ long terminal repeat (LTR) of these vectors or the human cytomegalovirus enhancer region (CMVe) inserted 5′ and 3′ of the retroviral LTRs. Furthermore, an extended AT stretch/attachment site (AT/att) of wild-type MLV was introduced into the vector. In the vector-producing cells, insertion of the CMVe and/or the 3pA resulted in a three- to fourfold-enhanced marker gene expression compared to the parental vector, whereas insertion of the AT/att gave a slight decrease in expression. The combination of all three modifications had no additional effects. In contrast, however, neomycin selection of infected cells revealed only a slight increase in virus titer with vectors carrying the 3pA modification; the titer was increased by 1 with vectors containing the extended AT/att, although the viral DNA copy numbers in infected cells were similar with both types of vectors. Thus, insufficient integration rather than insufficient reverse transcription and/or production of virus RNA is the major cause for the low titer obtained with the ProCon vectors. The combination of all three modifications resulted in a 2- to 3-log increase in the virus titer. These modifications result in expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors.

Comparative evaluation of preclinical in vivo models for the assessment of replicating retroviral vectors for the treatment of glioblastoma

Despite impressive improvements in neurosurgical techniques, radiation and chemotherapy during the past few years, little progress has been made in the treatment of malignant gliomas. Recently, the efficacy of suicide gene therapy based on replication-competent retroviral (RCR) vectors as delivery vehicles for the therapeutic gene has been described in the treatment of experimental cancer, including gliomas. In this study, we have thus critically evaluated a panel of human and rodent glioma/glioblastoma cell lines (U-87MG, U-118MG, LN-18, LN-229, 8-MG-BA, 42-MG-BA, A-172, T-98G, UVW, C6, 9L, G-26, GL-261, Tu-2449, Tu-9648) with respect to RCR virus vector spread, sensitivity towards the cytosine deaminase (CD)/5-flurocytosine (5-FC)/5-flurouracil (5-FU) suicide system, and orthotopic growth characteristics in mice to identify suitable preclinical animal models for the development of a glioblastoma gene therapy. Rapid virus spread was observed in eight out of nine human cell lines tested in vitro. As expected, only CD-expressing cells became sensitive to 5-FC, due to their ability to convert the prodrug in its toxic form, 5-FU. All LD50 values were within the range of concentrations obtained in human body fluids after conventional antifungal 5-FC administration. In addition, a significant bystander effect was observed in all human glioma cell lines tested. Injection of the RCR vector into pre-established orthotopic mouse tumor xenografts revealed substantial infection and virus spread of tumor tissue from most cell types.

Simian immunodeficiency virus vector pseudotypes differ in transduction efficiency and target cell specificity in brain

Lentiviral vectors have proven to be promising tools for transduction of brain cells in vivo and in vitro. In this study, we have examined the central nervous system (CNS) transduction efficiencies and patterns of a self-inactivating simian immunodeficiency virus (SIVmac)-derived lentiviral vector pseudotyped with glycoproteins from the vesicular stomatitis virus (VSV-G), the amphotropic murine leukemia virus (MLV4070Aenv), the lymphocytic choriomeningitis virus (LCMV-GP), the Ross River virus (RRV-GP) and the rabies virus (RV-G). All glycoproteins were efficiently incorporated into SIV virions, allowing efficient transduction of neuronal cell lines as well as of primary dissociated mouse brain cell cultures. After injection of highly concentrated vector stocks into the striatum of adult mice, quantitative analyses revealed high transduction efficiency with VSV-G pseudotypes, while LCMV-GP and RV-G pseudotypes exhibited moderate transduction efficiencies. MLV4070Aenv and RRV-GP pseudotypes, however, showed only weak levels of transduction after stereotactic injection into the brain. Regarding cell tropism in vivo, VSV-G-pseudotyped SIV vectors transduced neuronal as well as glial cells, whereas all other pseudotypes preferentially transduced neuroglial cells. In addition, we analyzed the influence of the central polypurine tract (cPPT) in context of the VSV-G-pseudotyped SIV transfer vector for infection of brain cells. Deletion of the cPPT sequence from the transfer vector decreased the in vivo transduction efficiency by fourfold, and, more importantly, this modification changed the transduction pattern, since these vectors were no longer able to infect neuronal cells in vivo. Vector injection into the brain did elicit a humoral immune response in the injected hemisphere; however, no gross signs of inflammation could be detected. Analysis of the biodistribution of the vector revealed that, besides the injected brain region, no vector-specific sequences could be detected in any of the organs evaluated. These data indicate SIV vectors as efficient gene delivery vehicles for the treatment of neurodegenerative diseases.

Expression and immunolocalisation of follicle-stimulating hormone receptors in gonads of newborn and adult female horses

In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animals age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.

Reconstituting retroviral (ReCon) vectors facilitating delivery of cytotoxic genes in cancer gene therapy approaches

We have previously described the generation of reconstituting retroviral (ReCon) vectors designed for cancer gene therapy using cytotoxic gene products. The unique vector structure with a promoter physically separated from the transgene allows generation of stable virus producer cells irrespective of the toxic gene. The mechanism of synthesis of DNA from retroviral RNA dictates that infection leads to the reconstitution of functional expression cassettes in the target cell.

Evaluation of a Gene-Directed Enzyme-Product Therapy (GDEPT) in Human Pancreatic Tumor Cells and Their Use as In Vivo Models for Pancreatic Cancer

Background Gene-directed enzyme prodrug therapy (GDEPT) is a two-step treatment protocol for solid tumors that involves the transfer of a gene encoding a prodrug-activating enzyme followed by administration of the inactive prodrug that is subsequently activated by the enzyme to its tumor toxic form. However, the establishment of such novel treatment regimes to combat pancreatic cancer requires defined and robust animal model systems. Methods Here, we comprehensively compared six human pancreatic cancer cell lines (PaCa-44, PANC-1, MIA PaCa-2, Hs-766T, Capan-2, and BxPc-3) in subcutaneous and orthotopical mouse models as well as in their susceptibility to different GDEPTs. Results Tumor uptake was 83% to 100% in the subcutaneous model and 60% to 100% in the orthotopical mouse model, except for Hs-766T cells, which did not grow orthotopically. Pathohistological analyses of the orthotopical models revealed an infiltrative growth of almost all tumors into the pancreas; however, the different cell lines gave rise to tumors with different morphological characteristics. All of the resultant tumors were positive for MUC-1 staining indicating their origin from glandular or ductal epithelium, but revealed scattered pan-cytokeratin staining. Transfer of the cytochrome P450 and cytosine deaminase suicide gene, respectively, into the pancreatic cancer cell lines using retroviral vector technology revealed high level infectibility of these cell lines and allowed the analysis of the sensitivity of these cells to the chemotherapeutic drugs ifosfamide and 5-fluorocytosine, respectively. Conclusion These data qualify the cell lines as part of valuable in vitro and in vivo models for the use in defined preclinical studies for pancreas tumor therapy.

Expression of ezrin and moesin in primary breast carcinoma and matched lymph node metastases

Ezrin, radixin, moesin (ERM) are important membrane-cytoskeletal crosslinkers and are suggested to play important role in cancer progression and metastasis. Even though ERM proteins were generally considered to be functionally redundant and the most studied was ezrin, recent studies highlight their distinct roles in metastatic process. Little information is available regarding the role of individual ERM proteins and their phosphorylated forms in human breast cancer. Our study is the first to examine expression of ezrin, moesin and their phosphorylated forms in primary breast tumors and matched lymph node metastases (LNMs) and their correlation with clinicopathological variables. A total of 88 primary breast cancer, 91 LNMs, 54 intraductal carcinoma and 26 normal adjacent breast tissue samples from tissue microarrays were studied. Expression was determined by immunohistochemistry, the intensity and number of positive cells was scored. Statistical analysis of protein expression and patients’ age, tumor grade and hormonal status was performed. No statistical significant difference was found in ezrin, moesin, p-ezrinTyr353 and pan-p-ezrinThr567/radixinThr564/moesinThr558 expression between primary tumors and LNMs. Even though it was not significant, moesin expression varied between primary tumors, intraductal carcinoma, normal breast adjacent tissue and LNMs. A significant positive correlation between moesin and tumor grade has been proven. Even though primary tumors and matched LNMs did not show different expression patterns, moesin correlated significantly with higher tumor grade. Its positivity in intraductal carcinoma and normal breast tissue adjacent to cancer might indicate its role in tumor intiation/progression.