Efficient protection of microorganisms for delivery to the intestinal tract by cellulose sulphate encapsulation.

BACKGROUND: Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic microorganisms can improve health. However, orally delivered probiotic microorganisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic microorganisms are still not effective enough. RESULTS: We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS), that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for at least 4 h without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by cellulase at concentrations found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract. CONCLUSION: Taken together, the data suggests that CS microencapsulation can protect bacteria and yeasts from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.

Recent developments linking retroviruses to human breast cancer: infectious agent, enemy within or both?

Evidence is accumulating that one or more beta-retrovirus is associated with human breast cancer. Retroviruses can exist as an infectious (exogenous) virus or as a part of the genetic information of cells due to germline integration (endogenous). An exogenous virus with a genome that is highly homologous to mouse mammary tumour virus is gaining acceptance as possibly being associated with human breast cancer, and recently furnished evidence is discussed in this article, as is the evidence for involvement of an endogenous human beta-retrovirus, HERV-K. Modes of interaction are also reviewed and linkages to the APOBEC3 family are suggested.

Revisiting a role for a mammary tumor retrovirus in human breast cancer.

There remains great controversy as to whether mouse mammary tumor virus (MMTV), the etiological agent of mammary cancer in mice, or a closely related human retrovirus, plays a role in the development of breast cancer in humans. On one hand, retroviruses such as human T-cell lymphotropic virus and human immunodeficiency virus (HIV) are known causative agents of cancer (in the case of HIV, albeit, indirectly), but attempts to associate other retroviruses with human cancers have been difficult. A recent, high profile, example has been the postulated involvement of another mouse virus, xenotropic murine leukemia virus-related virus, in human prostate cancer, which is now thought to be due to contamination. Here, we review some of the more recent evidence for and against the involvement of MMTV in human breast cancer and suggest future studies that may allow a definitive answer to this conundrum.

Long-Term Survival of Cellulose Sulphate-Encapsulated Cells and Metronomic Ifosfamide Control Tumour Growth in Pancreatic Cancer Models-A Prelude to Treating Solid Tumours Effectively in Pets and Humans.

BACKGROUND: The use of encapsulated cells for the in vivo delivery of biotherapeutics is a promising new technology to potentiate the effectiveness of cell-based therapies for veterinary and human application. One use of the technology is to locally activate chemotherapeutics to their short-lived highly active forms. We have previously shown that a stable clone of HEK293 cells overexpressing a cytochrome P450 enzyme that has been encapsulated in immunoprotective cellulose sulphate beads can be implanted near solid tumours in order to activate oxazaphosphorines such as ifosfamide and cyclophosphamide to the tumour-killing metabolite phosphoramide mustard. The efficacy of this approach has been shown in animal models as well as in human and canine clinical trials. In these previous studies, the oxazaphosphorine was only given twice. An analysis of the Kaplan-Meier plots of the results of the clinical trials suggest that repeated dosing might result in a significant clinical benefit. AIMS: In this study, we aimed to (i) demonstrate the stable long-term expression of cytochrome P450 from a characterized, transfected cell clone, as well as (ii) demonstrate that one implanted dose of these encapsulated cytochrome P450-expressing cells is capable of activating multiple doses of ifosfamide in animal models. METHODOLOGY: We initially used cell and molecular methods to show cell line stability over multiple passages, as well as chemical and biological function in vitro. This was followed by a demonstration that encapsulated HEK293 cells are capable of activating multiple doses of ifosfamide in a mouse model of pancreatic cancer without being killed by the chemotherapeutic. CONCLUSION: A single injection of encapsulated HEK293 cells followed by multiple rounds of ifosfamide administration results in repeated anti-tumour activity and halts tumour growth but, in the absence of a functioning immune system, does not cause tumour regression.

A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells.

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based applications for more effective and affordable therapies.

The 5' leader sequence of mouse mammary tumor virus enhances expression of the envelope and reporter genes.

Mouse mammary tumor virus (MMTV) is a complex betaretrovirus, which utilizes a Rev-like auxiliary protein Rem to export the unspliced viral RNA from the nucleus. MMTV env mRNA appears to be exported via a distinct, Rem-independent, mechanism. Here, we analysed the effect of an extensively folded region coinciding with the 5' leader sequence on env gene expression. We found that the presence of the 5' leader stimulates expression of the envelope protein. Enhanced Env production was accompanied by increased cytoplasmic levels of env mRNA. The 5' leader promotes nucleocytoplasmic translocation and increases stability of env mRNA. The region responsible for this effect was mapped to the distal part of the 5' leader. Furthermore, the 5' leader inserted in the sense orientation into a heterologous luciferase expression construct increased luciferase activity.

The Role of a Betaretrovirus in Human Breast Cancer: Enveloping a Conundrum.

Most of the evidence that a human betaretrovirus (HBRV/HMTV) highly related to mouse mammary tumour virus (MMTV) has an etiological role in breast cancer has been summarized in a recent comprehensive Special Issue of "Viruses" entitled "Human Betaretrovirus (HBRV) and Related Diseases". Shortly after publication of this special issue, a detailed analysis of aligned env sequences was published and concluded that (i) MMTV and HBRV/HMTV cannot be distinguished on the basis of aligned env sequences and (ii) more sequence data covering the full-length env or HBRV/HMTV genomes from multiple isolates is needed. Although productive infection of human cells by MMTV (and presumably HBRV/HMTV) has been shown, it is imperative that the receptor(s) enabling HBRV/HMTV to infect human cells are defined. Moreover, there is currently no compelling data for common integration sites, in contrast to MMTV induced mammary tumorigenesis in mice, suggesting that other mechanisms of tumorigenesis are associated with HBRV/HMTV infection. These issues need to be resolved before a clear link between MMTV/HBRV/HMTV and human breast cancer can be concluded.

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 LD(50) 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.

Human endogenous retroviral long terminal repeat sequences as cell type-specific promoters in retroviral vectors.

The human genome contains more than half a million human endogenous retrovirus (HERV) long terminal repeats (LTRs) that can be regarded as mobile regulatory modules. Many of these HERV LTRs have been recruited during evolution as transcriptional control elements for cellular gene expression. We have cloned LTR sequences from two HERV families, HERV-H and HERV-L, differing widely in their activity and tissue specificity into a murine leukemia virus (MLV)-based promoter conversion vector (ProCon). Various human cell lines were infected with the HERV-MLV hybrid vectors, and cell type-specific expression of the reporter gene was compared with the promoter specificity of the corresponding HERV LTRs in transient-transfection assays. Transcription start site analysis of HERV-MLV hybrid vectors revealed preferential use of the HERV promoter initiation site. Our data show that HERV LTRs function in the context of retroviral vectors in certain cell types and have the potential to be useful as cell type-specific promoters in vector construction.

Identification of the Rem-responsive element of mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) has previously been shown to encode a functional homolog of the human immunodeficiency virus-1 (HIV-1) nuclear export protein Rev, termed Rem. Here, we show that deletion of the rem gene from a MMTV molecular clone interfered with the nucleo-cytoplasmic transport of genomic length viral mRNA and resulted in a loss of viral capsid (Gag) protein production. Interestingly, nuclear export of single-spliced env mRNA was only moderately affected, suggesting that this transcript is, at least to some extent, transported via a distinct, Rem-independent export mechanism. To identify and characterize a cis-acting RNA element required for Rem responsiveness (RmRE), extensive computational and functional analyses were performed. By these means a region of 490 nt corresponding to positions nt 8517-nt 9006 in the MMTV reference strain was identified as RmRE. Deletion of this fragment, which spans the env-U3 junction region, abolished Gag expression. Furthermore, insertion of this sequence into a heterologous HIV-1-based reporter construct restored, in the presence of Rem, HIV-1 Gag expression to levels determined for the Rev/RRE export system. These results clearly demonstrate that the identified region, whose geometry resembles that of other retroviral-responsive elements, is capable to functionally substitute, in the presence of Rem, for Rev/RRE and thus provide unequivocal evidence that MMTV is a complex retrovirus.

Therapeutic application of cell microencapsulation in cancer.

Although cell encapsulation technologies were originally developed for the treatment of acquired and genetic diseases such as diabetes, they can also be applied to the treatment of a variety of solid tumours. There are a number of strategies aimed at treating tumours with encapsulated cells and most of these are reviewed in this chapter. Many of these strategies have shown promise in preclinical studies and clinical trials.

Semipermeable Cellulose Beads Allow Selective and Continuous Release of Small Extracellular Vesicles (sEV) From Encapsulated Cells.

The clinical benefit of therapies using Mesenchymal Stem Cells (MSCs) is attributable to their pleiotropic effect over cells and tissues, mainly through their secretome. This paracrine effect is mediated by secreted growth factors and extracellular vesicles (EV) including small EV (sEV). sEV are extra-cellular, membrane encompassed vesicles of 40 to 200 nm diameter that can trigger and signal many cellular responses depending on their cargo protein and nucleic acid repertoire. sEV are purified from cell culture conditioned media using several kits and protocols available that can be tedious and time-consuming, involving sequences of ultracentrifugations and density gradient separations, making their production a major challenge under Good Manufacturing Practices (GMP) conditions. We have developed a method to efficiently enrich cell culture media with high concentrations of sEV by encapsulating cells in semipermeable cellulose beads that allows selectively the release of small particles while offering a 3D culture condition. This method is based on the pore size of the capsules, allowing the release of particles of ≤ 200 nm including sEV. As a proof-of-principle, MSCs were encapsulated and their sEV release rate (sEV-Cap) was monitored throughout the culture and compared to sEV isolated from 2D seeded cells (sEV-2D) by repetitive ultracentrifugation cycles or a commercial kit. The isolated sEV expressed CD63, CD9, and CD81 as confirmed by flow cytometry analysis. Under transmission electron microscopy (TEM), they displayed the similar rounded morphology as sEV-2D. Their corresponding diameter size was validated by nanoparticle tracking analysis (NTA). Interestingly, sEV-Cap retained the expected biological activities of MSCs, including a pro-angiogenic effect over endothelial cells, neuritic outgrowth stimulation in hippocampal neurons and immunosuppression of T cells in vitro. Here, we successfully present a novel, cost, and time-saving method to generate sEV from encapsulated MSCs. Future applications include using encapsulated cells as a retrievable delivery device that can interact with the host niche by releasing active agents in vivo, including sEV, growth factors, hormones, and small molecules, while avoiding cell clearance, and the negative side-effect of releasing undesired components including apoptotic bodies. Finally, particles produced following the encapsulation protocol display beneficial features for their use as drug-loaded delivery vehicles.

Influence of vector design and host cell on the mechanism of recombination and emergence of mutant subpopulations of replicating retroviral vectors.

BACKGROUND: The recent advent of murine leukaemia virus (MLV)-based replication-competent retroviral (RCR) vector technology has provided exciting new tools for gene delivery, albeit the advances in vector efficiency which have been realized are also accompanied by a set of fresh challenges. The expression of additional transgene sequences, for example, increases the length of the viral genome, which can lead to reductions in replication efficiency and in turn to vector genome instability. This necessitates efforts to analyse the rate and mechanism of recombinant emergence during the replication of such vectors to provide data which should contribute to improvements in RCR vector design. RESULTS: In this study, we have performed detailed molecular analyses on packaged vector genomes and proviral DNA following propagation of MLV-based RCR vectors both in cell culture and in pre-formed subcutaneous tumours in vivo. The effects of strain of MLV, transgene position and host cell type on the rate of emergence of vector recombinants were quantitatively analysed by applying real-time PCR and real-time RT-PCR assays. Individual mutants were further characterized by PCR, and nucleotide sequence and structural motifs associated with these mutants were determined by sequencing. Our data indicate that virus strain, vector design and host cell influence the rate of emergence of predominating vector mutants, but not the underlying recombination mechanisms in vitro. In contrast, however, differences in the RNA secondary structural motifs associated with sequenced mutants emerging in cell culture and in solid tumours in vivo were observed. CONCLUSION: Our data provide further evidence that MLV-based RCR vectors based on the Moloney strain of MLV and containing the transgene cassette in the 3' UTR region are superior to those based on Akv-MLV and/or containing the transgene cassette in the U3 region of the LTR. The observed discrepancies between the data obtained in solid tumours in vivo and our own and previously published data from infected cells in vitro demonstrates the importance of evaluating vectors designed for use in cancer gene therapy in vivo as well as in vitro.

Transgene expression facilitated by the v-src splice acceptor can impair replication kinetics and lead to genomic instability of Rous sarcoma virus-based vectors.

Rous sarcoma virus (RSV) can be used for the simple generation of high-titer replication-competent retroviral (RCR) vectors. Retroviruses undergo frequent genomic recombination, however, and vectors with reduced replication kinetics are rapidly overgrown by mutant forms. Vector design is hence critical to vector efficacy. In this study, two different designs of RSV-based RCR vectors were evaluated. Vectors in which transgene expression was facilitated by the v-src splice acceptor were revealed to have greatly reduced replication kinetics and genomic stability in comparison to vectors in which transgene expression was mediated by an internal ribosome entry site in the 3' untranslated region.

Mouse mammary tumor virus integration site selection in human and mouse genomes.

Based on integration site preferences, retroviruses can be placed into three groups. Viruses that comprise the first group, murine leukemia virus and foamy virus, integrate preferentially near transcription start sites. The second group, notably human immunodeficiency virus and simian immunodeficiency virus, preferentially targets transcription units. Avian sarcoma-leukosis virus (ASLV) and human T-cell leukemia virus (HTLV), forming the third group, show little preference for any genomic feature. We have previously shown that some human cells sustain mouse mammary tumor virus (MMTV) infection; therefore, we infected a susceptible human breast cell line, Hs578T, and, without introducing a species-specific bias, compared the MMTV integration profile to those of other retroviruses. Additionally, we infected a mouse cell line, NMuMG, and thus we could compare MMTV integration site selection in human and mouse cells. In total, we examined 468 unique MMTV integration sites. Irrespective of whether human or mouse cells were infected, no integration bias favoring transcription start sites was detected, a profile that is reminiscent of that of ASLV and HTLV. However, in contrast to ASLV and HTLV, not even a modest tendency in favor of integration within genes was observed. Similarly, repetitive sequences and genes that are frequently tagged by MMTV in mammary tumors were not preferentially targeted in cell culture either in mouse or in human cells; hence, we conclude that MMTV displays the most random dispersion of integration sites among retroviruses determined so far.

Association of glycosylphosphatidylinositol-anchored protein with retroviral particles.

We describe for the first time the association of glycosylphosphatidylinositol (GPI) -anchored proteins with retroviral and lentiviral particles, similar to a process well established for cells, termed "painting." The aim of the study was to assess the feasibility of modification of retroviral vectors by exogenous addition of recombinant protein, removing the need for genetic engineering of virus producer cell lines. The recombinant GPI protein CD59his was purified via fast protein liquid chromatography and associated with concentrated virus stock in a controlled incubation procedure. Reaction mixtures were purified in order to remove nonassociated GPI protein and endogenous protein. Analysis of samples by immunoblotting revealed that CD59his was only detectable in the presence of viral particles. From this, we conclude that CD59his could be stably associated with retroviral particles. In addition, we demonstrated by flow cytometry that virus particles remain infectious after these procedures. As well as suggesting a novel possibility for interaction between enveloped virus and host, we believe that the stable association of recombinant GPI proteins to retroviral particles can be developed into an important tool for both research and clinical applications, especially in the fields of gene therapy and vaccine development.

Molecularly characterised xenograft tumour mouse models: valuable tools for evaluation of new therapeutic strategies for secondary liver cancers.

To develop and evaluate new therapeutic strategies for the treatment of human cancers, well-characterised preclinical model systems are a prerequisite. To this aim, we have established xenotransplantation mouse models and corresponding cell cultures from surgically obtained secondary human liver tumours. Established xenograft tumours were patho- and immunohistologically characterised, and expression levels of cancer-relevant genes were quantified in paired original and xenograft tumours and the derivative cell cultures applying RT-PCR-based array technology. Most of the characteristic morphological and immunohistochemical features of the original tumours were shown to be maintained. No differences were found concerning expression of genes involved in cell cycle regulation and oncogenesis. Interestingly, cytokine and matrix metalloproteinase encoding genes appeared to be expressed differentially. Thus, the established models are closely reflecting pathohistological and molecular characteristics of the selected human tumours and may therefore provide useful tools for preclinical analyses of new antitumour strategies in vivo.

Quantification and characterization of autotransduction in retroviral vector producer cells.

Gene therapy has evolved into a tempting strategy for the management of cancer and other life-threatening diseases. Various approaches employ retroviral vectors to deliver the therapeutic gene. The profound knowledge about retrovirus biology allows the generation of increasingly advanced vector systems as well as an accurate assessment and management of potential safety risks. This study focuses on the genetic stability of retrovirus producer cells as a basic safety requirement and its compromise by autotransduction. It has been shown previously that protection of retroviral packaging systems by superinfection interference is not guaranteed. The current study provides insight into the extent of autotransduction and the time point at which it occurs, and examines strategies to antagonize it. Therefore, a reconstituting vector system was used that obviates transgene expression in virus producer cells by physically separating transgene and promoter. Just on infection two functional expression cassettes are reconstituted, causing highly efficient transgene expression in transduced cells. Equipped with an enhanced green fluorescent protein-encoding gene, this vector allowed accurate quantification of autotransduced cells, which were then isolated by fluorescence-activated cell sorting and further characterized. Sequencing of recloned integrated vector copies demonstrated that high transgene expression levels were strictly associated with the presence of reverse-transcribed vector copies. Envelope protein expression levels, however, were found to be equal in autotransduced and noninfected virus producer cells. Finally, the occurrence of autotransduction could be assigned to an early time point after transfection and was successfully blocked by azidothymidine treatment, yielding a stable and homogeneous population of noninfected retrovirus producer cells.

Mouse mammary tumor virus promoter-containing retroviral promoter conversion vectors for gene-directed enzyme prodrug therapy are functional in vitro and in vivo.

Gene directed-enzyme prodrug therapy (GDEPT) is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC(50) values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.