In-depth characterization of a new patient-derived xenograft model for metaplastic breast carcinoma to identify viable biologic targets and patterns of matrix evolution within rare tumor types.

Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype with rapid growth, high rates of metastasis, recurrence and drug resistance, and diverse molecular and histological heterogeneity. Patient-derived xenografts (PDXs) provide a translational tool and physiologically relevant system to evaluate tumor biology of rare subtypes. Here, we provide an in-depth comprehensive characterization of a new PDX model for MBC, TU-BcX-4IC. TU-BcX-4IC is a clinically aggressive tumor exhibiting rapid growth in vivo, spontaneous metastases, and elevated levels of cell-free DNA and circulating tumor cell DNA. Relative chemosensitivity of primary cells derived from TU-BcX-4IC was performed using the National Cancer Institute (NCI) oncology drug set, crystal violet staining, and cytotoxic live/dead immunofluorescence stains in adherent and organoid culture conditions. We employed novel spheroid/organoid incubation methods (Pu·MA system) to demonstrate that TU-BcX-4IC is resistant to paclitaxel. An innovative physiologically relevant system using human adipose tissue was used to evaluate presence of cancer stem cell-like populations ex vivo. Tissue decellularization, cryogenic-scanning electron microscopy imaging and rheometry revealed consistent matrix architecture and stiffness were consistent despite serial transplantation. Matrix-associated gene pathways were essentially unchanged with serial passages, as determined by qPCR and RNA sequencing, suggesting utility of decellularized PDXs for in vitro screens. We determined type V collagen to be present throughout all serial passage of TU-BcX-4IC tumor, suggesting it is required for tumor maintenance and is a potential viable target for MBC. In this study we introduce an innovative and translational model system to study cell-matrix interactions in rare cancer types using higher passage PDX tissue.

Gene transfer to the rhesus monkey brain using SV40-derived vectors is durable and safe.

Gene transfer to central nervous system (CNS) has been approached using various vectors. Recombinant SV40-derived vectors (rSV40s) transduce human neurons and microglia effectively in vitro and in rodent brains in vivo, so we tested rSV40s gene transfer to rhesus monkey CNS in vivo, to characterize the distribution, duration and safety of such gene delivery. We used rSV40s carrying HIV-1 RevM10 with a carboxyl-terminal AU1 epitope tag as a marker, and others with the antioxidant enzymes, Cu/Zn superoxide dismutase and glutathione peroxidase. Vectors were injected stereotaxically into the caudate nucleus. Transgene expression was studied at 1 and 6 months by immunostaining serial brain sections. After intraparenchymal administration, numerous transgene-expressing cells were seen, with a longitudinal extent of 20 mm. In neurons and, more rarely, microglial cells, transgene expression remained strong throughout the 6-month study period. Astrocytes and oligodendroglia were not transduced. No evidence of inflammation or tissue damage was observed. SV40-derived vectors may thus be useful for long-term gene expression in the monkey brain and, potentially, in the human brain.

Stromal stem cells from adipose tissue and bone marrow of age-matched female donors display distinct immunophenotypic profiles.

Adipose tissue is composed of lipid-filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose-derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA-abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7-fold vs. 2.85-fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT-PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage-specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine.

Reduction in SIV replication in rhesus macaques infused with autologous lymphocytes engineered with antiviral genes.

Simian immunodeficiency virus (SIV) infection of nonhuman primates is one of the most relevant animals models of HIV infection in humans. To test a potential anti-HIV gene therapy strategy in this model, CD4-enriched lymphocytes from three rhesus macaques were subjected to retrovirally mediated gene transfer with a vector expressing an antisense tat/rev gene. This group of animals and three control macaques were subsequently infected with SIVmac239. Blood and lymph nodes from all macaques were sampled for more than a year to monitor the progress of infection. Although all animals became infected, the animals that received the lymphocytes engineered with the antisense vector demonstrated a significant reduction in viral load in both peripheral blood and lymph nodes, had sustained numbers of CD4+ cells, and exhibited little disruption of lymph node architecture.

Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryos.

A series of experiments was performed to determine the dynamics of pronuclear development as well as the efficiency of either adenovirus-associated (AAV) or lentivirus-derived vectors to introduce a green fluorescent protein (GFP) reporter gene into rhesus macaque (Macaca mulatta) embryos. Assessment of pronuclear development at various times after fertilization revealed that the appearance of pronuclei was determined by the presence of the first and the timing of the second polar body. The dynamics of pronuclear formation was a significant determinant of whether an oocyte reached the blastocyst stage, however, when the percentage of blastocysts were based on the number of zygotes, the timing of the appearance of polar bodies did not appear to have any effect on subsequent development. Injection of different AAV-derived vectors showed that the serotype of the vector did not affect development or the proportion of transgenic embryos. Moreover, all putative transgenic embryos proved to be expression mosaics. Injection of embryos with lentiviral vectors showed that timing of injection (before or after fertilization) had no effect on subsequent transgene expression, but that the type of reporter gene determined post-injection development and rate of transgenesis. The transfer of embryos following injection of a lentiviral vector into three recipients resulted in one pregnancy which was lost during the second trimester. Analysis of fetal tissues showed ubiquitous presence of the transgene and GFP expression in all tissues examined. These results show that lentivirus-derived vectors can efficiently transform rhesus embryos and are suitable for the generation of transgenic rhesus monkeys.

Human cytomegalovirus infection of human adipose-derived stromal/stem cells restricts differentiation along the adipogenic lineage.

Human adipose-derived stromal/stem cells (ASCs) display potential to be used in regenerative stem cell therapies and as treatments for inflammatory and autoimmune disorders. Despite promising use of ASCs as therapeutics, little is known about their susceptibility to infectious agents. In this study, we demonstrate that ASCs are highly susceptible to human cytomegalovirus (HCMV) infection and permissive for replication leading to release of infectious virions. Additionally, many basic ASC functions are inhibited during HCMV infection, such as differentiation and immunomodulatory potential. To our knowledge this is the first study examining potential adverse effects of HCMV infection on ASC biology. Our results suggest, that an active HCMV infection during ASC therapy may result in a poor clinical outcome due to interference by the virus.

Analysis of gene transfer efficiency of retrovirus producer cell transplantation for in situ gene transfer to hematopoietic cells.

OBJECTIVE: The aim of this study was to assess the gene transfer efficiency of an in situ administration protocol for hematopoietic stem/progenitor cells in the rhesus macaque (Macaca mulatta) animal model. MATERIALS AND METHODS: Moloney murine leukemia virus amphotropic vector producer cells (1--2 x 10(8) cells/animal) were transplanted into the femoral bone marrow cavities of six macaques. To determine if the levels of gene transfer could be increased, a second injection at the same dose of producer cells was performed into the iliac crest in three of the six macaques. RESULTS: We demonstrated that 0.02-0.1% of peripheral blood mononuclear cells contained the vector transgene for up to 12 months following the initial administration of producer cells. Hematopoietic progenitor cell assays indicated that the neomycin phosphotransferase gene was detected in 10--30% of progenitor cell colonies. A humoral immune response directed toward viral particles was demonstrated in all animals. Additionally, we demonstrated that an increase in the levels of transduced cells, up to 1% of circulating peripheral blood mononuclear cells and granulocytes, contain the transgene following producer cell readministration. CONCLUSIONS: These data demonstrate the successful in situ gene transfer to hematopoietic stem/progenitor cells and circulating peripheral blood mononuclear cells that persists as long as 12 months postinjection, in the absence of any preconditioning.

Targeted transduction of CD34+ cells by transdominant negative Rev-expressing retrovirus yields partial anti-HIV protection of progeny macrophages.

Congenitally acquired HIV infection may be uniquely suited to treatment via genetic engineering of CD34+ hematopoietic stem/progenitor cells. However, current technologies yield only a small percentage of mature cells that carry the inserted genes, and expression is frequently suppressed. Since clinical trials employing these methodologies have been proposed for anti-HIV gene therapy of HIV-infected children, we wished to assess, by in vitro modeling, the expected limits of transduction efficiency, expression, and antiviral activity using currently available methods. We measured retrovirus-mediated transduction in cord blood progenitors and their in vitro-derived progeny macrophages by Mo-MuLV vectors expressing a transdominant negative Rev (RevTD). CFU-GM transduction efficiency ranged from 7 to 85%, with an average of 28%. Semiquantitative DNA PCR demonstrated < or =100 vector sequence copies per 1000 cells in monocyte/macrophage cultures, which were grown without selection to better model in vivo conditions. When challenged with the macrophagetropic HIV-1BaL isolate, cultured macrophages from mock-transduced CFU-GM colonies supported infection in eight of eight experimental cultures, control LXSN-transduced progenitors supported infection in six of eight cultures, while macrophages derived from RevTD-transduced CFU-GM colonies supported infection in four of eight cultures. Although these results support the ability of neo(r) retroviral vectors containing RevTD to inhibit HIV replication, they indicate that further optimization of transduction efficiency and sustained expression will be required for effective anti-HIV protection in vivo.

Optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer.

Gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. Optimized HIV-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. However, the use of HIV-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. We report that vectors based on the non-primate equine infectious anemia virus (EIAV) transduce a variety of human hematopoietic cell lines and primary blood cells. To investigate optimization of gene expression in hematopoietic cells, we compared a variety of post-transcriptional elements and promoters in the context of EIAV vectors. We observed cell specific increase in the number of transgene expressing cells with the different post-transcriptional elements, whereas the use of elongation factor alpha 1 (EFalpha1) promoter resulted in significant increases in both the number of transgene expressing cells and the level of transgene protein in all cell types tested. We then demonstrate increased transduction of hematopoietic cells using a second-generation EIAV vector containing a self-inactivating EIAV LTR and the EIAV central polypurine tract (cppt). These data suggest that optimized EIAV vectors may be a suitable alternative to HIV-1 vectors for use in hematopoietic gene therapy.

Gene therapy for infectious diseases.

Gene therapy is being investigated as an alternative treatment for a wide range of infectious diseases that are not amenable to standard clinical management. Approaches to gene therapy for infectious diseases can be divided into three broad categories: (i) gene therapies based on nucleic acid moieties, including antisense DNA or RNA, RNA decoys, and catalytic RNA moieties (ribozymes); (ii) protein approaches such as transdominant negative proteins and single-chain antibodies; and (iii) immunotherapeutic approaches involving genetic vaccines or pathogen-specific lymphocytes. It is further possible that combinations of the aforementioned approaches will be used simultaneously to inhibit multiple stages of the life cycle of the infectious agent.

Transient expression of a p58 protein kinase cDNA enhances mammalian glycosyltransferase activity.

The effect of expression of a p58 protein kinase on mammalian beta-1,4 galactosyltransferase enzyme activity was examined in vitro and in vivo. We found that p58 protein kinase expression enhanced galactosyltransferase enzyme activity approximately three-fold in vivo when compared to reporter gene activity. Galactosyltransferase enzyme activity was also substantially reduced in vitro when dephosphorylated, or when p58 specific antibodies were used to inhibit kinase activity. These results suggest that galactosyltransferase activity is influenced by phosphorylation, and that the p58 protein kinase may mediate this effect.

Targeted delivery of antisense oligonucleotides by molecular conjugates.

Antisense oligonucleotides efficiently inhibit gene expression in vitro; however, the successful therapeutic application of this technology in vivo will require the development of improved delivery systems. In this report we describe a technique that efficiently delivers antisense oligonucleotides into cells using molecular conjugates. This technique, which was initially developed for the delivery of eukaryotic genes, is based on the construction of DNA-protein complexes that are recognized by the liver-specific asialoglycoprotein receptor. Binding of poly(L-lysine)-asialoorosomucoid (AsOR) protein conjugates with phosphorothioate antisense oligonucleotides to chloramphenicol acetyltransferase (CAT) led to the formation of 50- to 150-nm toroids. Exposure of the antisense molecular complexes (3 microM oligonucleotide) to NIH 3T3 cells genetically modified to express both the AsOR receptor and CAT, inhibited CAT expression by 54%, which was completely blocked by excess AsOR. Equivalent inhibition of CAT activity with purified oligonucleotide alone was observed at a 30 microM concentration. Furthermore, examination of the cells using indirect immunofluorescence for the presence of CAT protein showed 28% of cells exposed to the molecular conjugates lacked any detectable CAT enzyme. Cells exposed to oligonucleotide alone showed a highly variable staining pattern, and only a few of the cells were completely void of CAT protein. Together these data demonstrate that molecular conjugates provide a highly specific and efficient system for the delivery of antisense oligonucleotides.

A dominant negative mutation in two proteins created by ectopic expression of an AU-rich 3' untranslated region.

We have found that the ectopic expression of a specific gene's 3' untranslated region leads to the dose dependent loss, relative to gene copy number, of that specific mRNA and protein, as well as an associated protein, in a eukaryotic cell line. The loss of these proteins from the eukaryotic cell line also results in specific phenotypic changes in these cells, suggesting that we have created a dominant negative mutant. This gene's 3' untranslated region is known to contain numerous AU sequences, reminiscent of other eukaryotic genes whose expression may be regulated by these sequences. The apparent control of gene expression by a truncated 3' untranslated region sequence provides further evidence supporting the regulatory function of these regions. The resulting decrease in steady-state mRNA levels by the overexpression of a portion of that gene's 3' untranslated region further suggests the possible existence of a factor(s) that may bind to this region, and thus regulate gene function via its mRNA. The use of a gene's 3' untranslated sequence to create a specific dominant negative mutation may also be applicable to other eukaryotic genes whose expression is controlled by similar regulatory sequences.

Efficient in vivo marking of primary CD4+ T lymphocytes in nonhuman primates using a gibbon ape leukemia virus-derived retroviral vector.

High efficiency retroviral-mediated gene transfer to rhesus CD4+ peripheral blood lymphocytes (PBL) was accomplished using an optimized transduction protocol using a gibbon ape leukemia virus (GaLV) envelope-containing packaging cell line PG13. Engineered CD4+ PBL were administered to three nonmyeloablated animals in three or four separate infusions over 9 months. Polymerase chain reaction (PCR) demonstrated in vivo reconstitution of the genetically engineered CD4+ PBL at levels between 1% and 10% of the circulating leukocytes. This level of gene marking indicates that up to 30% of endogenous circulating CD4+ cells can be genetically engineered. The high levels of marked lymphocytes persist for the first 3 weeks following reinfusion then decline to < or = 0.1% over the next 21 weeks. Lymph node (LN) biopsies were performed to determine if the engineered CD4+ lymphocytes could traffic to lymphoid tissues. Marked lymphocytes were detected in LN biopsies 100 days following reinfusion of the transduced cells. Expression of retroviral vector-derived sequences was detected by reverse transcriptase (RT)-PCR analysis from CD4-enriched lymphocytes that were activated by culturing in the presence of recombinant interleukin-2 (rlL-2). A humoral immune response to fetal bovine serum (FBS) was detected in all animals following the second administration of the culture expanded CD4+ lymphocytes. No antibody response was detected to the neomycin-resistance (Neo(R)) transgene, the murine retroviral group-specific antigen (gag), or GaLV envelope (env) proteins.

Increased expression of a 58-kDa protein kinase leads to changes in the CHO cell cycle.

We have isolated and characterized cDNA encoding a human 58-kDa protein kinase that is homologous to the cell division control (CDC) protein kinases. This protein kinase also contains a unique N-terminal domain that may potentially regulate its function. Due to its relatedness to p34CDC2, the human p58 cDNA was overexpressed in CHO cells to determine the effect on the cell cycle. Elevated expression of p58 in these cells resulted in prolonged late telophase and early G1 phase of the cell cycle. These p58 overexpressors showed a significantly increased frequency of tubulin midbodies as well as significant increases in mitotic abnormalities. Thus, proper regulation of p58 protein kinase is essential for normal cell cycle progression in these cells.

Comparison of gene transfer efficiencies and gene expression levels achieved with equine infectious anemia virus- and human immunodeficiency virus type 1-derived lentivirus vectors.

This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G1/S- and G2/M-arrested cells, and both had very low efficiencies of transduction into G0-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA than the EIAV-transduced cells. Measurements of vector-derived EGFP RNA half-lives were fourfold higher with the HIV vector than with the EIAV vector. Long-term culture of EIAV-transduced human cells showed a significant decrease in the number of cells expressing the transgene; however, no corresponding loss was found in EIAV-transduced equine cells. In contrast, only a moderate decrease in the number of transgene-expressing cells was seen with the HIV vectors. Taken together, these results demonstrate that the EIAV vectors transduced human cells with efficiencies similar to those of the HIV vectors. However, our data indicate that transgene expression from EIAV vectors is limited by the instability of vector-derived RNA transcripts and silencing of the EIAV vectors over time.

Analysis of gene transfer and expression in skeletal muscle using enhanced EIAV lentivirus vectors.

Skeletal muscle is an attractive target tissue for gene therapy involving both muscle and nonmuscle disorders. HIV-1-based vectors transduce mature skeletal muscle; however, the use of these vectors for human gene therapy may be limited by biosafety concerns. In this study, we investigated gene transfer using lentivirus vectors based on the equine infectious anemia virus (EIAV) in skeletal muscle in vitro and in vivo. EIAV vectors transduce proliferating and differentiating C2C12 mouse muscle cells; furthermore, the addition of the woodchuck hepatitis posttranscriptional element to EIAV vectors markedly increases gene expression in these cells. A single injection of EIAV vectors into skeletal muscle of adult mice led to detectable gene marking and gene expression for the duration of the 3-month study. Use of a second-generation EIAV self-inactivating vector (E-SIN) increased transduction in muscle cells in vitro, and injection of E-SIN vectors into skeletal muscle resulted in increased gene marking and gene expression compared to first-generation EIAV vectors.

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

Lentiviral vector gene transfer into fetal rhesus monkeys (Macaca mulatta): lung-targeting approaches.

We previously reported the efficiency of gene transfer in fetal monkeys using retroviral vectors and an intraperitoneal (IP) approach. Here, we explored intrapulmonary administration to determine whether gene transfer can be limited to the developing lung. The HIV-1-derived lentiviral vector (VSV-G pseudotyped; 1 x 10(7) infectious particles/fetus), using the enhanced green fluorescent protein (EGFP) as a reporter, was directly injected into fetal lung with ultrasound guidance (n=4; 55 or 70 days gestation; term 165+/-10 days). Fetuses were monitored sonographically, fetal/maternal blood samples collected during gestation, and four of four healthy newborns were delivered at term. All lung lobes were positive for the transgene (< or = 1%) when assessed by PCR, and transgene expression was observed by direct fluorescence microscopy and flow cytometry. The results of this study show the following: (1) successful gene transfer in fetal monkeys using an intrapulmonary approach; (2) less transduction of non-pulmonary tissues with gene transfer at 70 days gestation compared with 55 days gestation or use of an IP approach; (3) that the pulmonary epithelium was EGFP-positive by immunohistochemistry; and (4) no evidence of transplacental transport of vector sequences or antibody responses in the dams. The results of these investigations indicate the efficiency of fetal gene transfer by intrapulmonary delivery, and emphasize the importance of the fetal monkey as a preclinical model system for exploring in utero genetic treatment strategies for pulmonary disorders.

Development of improved adenosine deaminase retroviral vectors.

A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a "simplified" vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA-) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA- severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.