Transcriptional targeting of acute hypoxia in the tumour stroma is a novel and viable strategy for cancer gene therapy.

Deregulated tumour growth and neovascularization result in an inadequate tumour blood supply, leading to areas of chronic hypoxia and necrosis. Irregular vascular structure and abnormal tumour physiology also cause erratic blood flow in tumour vessels. We reasoned that tumour stroma, including vascular endothelial cells, would consequently experience transient hypoxia that may allow transcriptional targeting as part of an antivascular gene therapy approach to cancer. To exploit hypoxia for transcriptional regulation, retroviral vectors were generated with modified LTRs: a 6-mer of hypoxia response elements in place of the viral enhancer produced near wild-type levels of expression in hypoxia but was functionally inert in normoxia. In a tumour xenograft model, expression was mainly around areas of necrosis, which were shown to be hypoxic; no expression was detected in tumour stroma. Time-course experiments in vitro demonstrated that expression was transient in response to a hypoxic episode, such that a reporter gene would be insensitive to acute hypoxia in vivo. In contrast, a significant therapeutic effect was seen upon ganciclovir administration with a vector expressing thymidine kinase (TK) in the tumour stroma. Expression of TK was more effective when targeted to acute hypoxia in the stroma compared to chronic hypoxia in the poorly vascularized regions of the tumour cell compartment. The data presented here are evidence that hypoxia in the stromal compartment does occur and that transient hypoxia constitutes a valid therapeutic target.

Retroviral hybrid LTR vector strategy: functional analysis of LTR elements and generation of endothelial cell specificity.

Transcriptional targeting is an important aspect of developing gene therapy vectors in order to restrict transgene expression to selected target cells. One approach, when using retroviral vectors, is to replace viral transcriptional control elements within the long terminal repeat (LTR) with sequences imparting the desired specificity. We have developed such hybrid LTR retroviruses, incorporating sequences from each of the human promoters for flt-1, ICAM-2 and KDR, as part of our antivascular cancer gene therapy strategy targeting tumour endothelial cells. The chosen fragments were used to replace the enhancer or combined enhancer and proximal promoter regions of the viral LTR. All showed activity in primary human breast microvascular endothelial cells, with viruses incorporating ICAM-2 sequences exhibiting the greatest specificity versus nonendothelial cells in vitro and a marked alteration of specificity towards endothelial cells in a subcutaneous xenograft model in vivo. Moreover, our study documents the effect of enhancer and/or proximal promoter deletion on LTR activity and reports that differential dependence in different cell lines can give the false impression of specificity if experiments are not adequately controlled. This finding also has implications for other retroviral vector designs seeking to provide transcriptional specificity and for their safety with respect to prevention of gene activation at sites of proviral integration.

Reduced growth in response to ganciclovir treatment of subcutaneous xenografts expressing HSV-tk in the vascular compartment.

Using a recombinant retrovirus with ecotropic envelope we have achieved high efficiency of transduction of endothelial cells in the vasculature of subcutaneous xenografts arising from the co-injection of tumour cells and irradiated virus producers. We have used this experimental system to assess the efficacy of the herpes simplex virus-thymidine kinase (HSV-tk)/ganciclovir (GCV) prodrug activation system in anti-vascular therapy. Treatment of KSY-1 xenografts with HSV-tk transduction in the vascular compartment with the S-phase-dependent drug GCV resulted in extensive haemorrhagic necrosis, indicative of vascular damage. Therapeutic potential in tumours with transduced endothelial cells comprising 5% or less of the total tumour mass was similar to that of tumours with HSV-tk expression in over 46% of tumour cells. GCV treatment of animals bearing MDA-MB-361 breast carcinoma, SW620 and CACO2 colon carcinomas with HSV-tk expression in the vascular compartment also resulted in reduced tumour growth. We conclude that HSV-tk/GCV prodrug activation is an effective strategy for eradicating tumour vasculature, and that direct targeting of proliferating endothelial cells in established vasculature results in reduced tumour growth. The therapeutic potential observed with the slow-growing CACO2 colon and MDA-MB-361 breast carcinomas supports the notion that anti-vascular therapy targeted at proliferating endothelium is likely to prove efficacious in human cancers that generally grow at a lower rate than experimental tumours.

Generation of a high titre retroviral vector for endothelial cell-specific gene expression in vivo.

Tumour growth is dependent upon a blood supply and is associated with the switch to the angiogenic phenotype. We are developing strategies for targeting gene expression to endothelial cells in the tumour vasculature. Recombinant retroviruses have been generated that incorporate regulatory sequences of the prepro-endothelin-1 (ppET1) promoter. Following reverse transcription and integration these modifications are duplicated in the proviral 5' LTR for transcription of the internal beta-galactosidase reporter gene. The titres and endothelial specificity of retroviral vectors harbouring different modifications have been analysed. In the optimal strategy, replacing the MLV enhancer with ppET1 promoter sequences containing the GATA and AP1 elements whilst maintaining sequences from the viral promoter resulted in endothelial cell-specific expression of the reporter gene, and viral titres comparable to those of the unmodified vector. A panel of endothelial and non-endothelial cells infected with the modified virus from a high titre producer clone showed a pattern of expression consistent with the activity of the endogenous ppET1 promoter. The modified LTR retained specificity in vivo, in subcutaneous tumours arising from the co-injection of tumour cells and irradiated virus producer cells. This simple model achieves high efficiency of transduction and can be used routinely for the screening of targeted retroviral vectors. Gene Therapy (2000) 7, 368-376.

Emphysematous cholecystitis in a Siberian husky.

A 6-year-old, intact male Siberian husky was evaluated for a 24-hour history of vomiting and lethargy. Diagnosis of emphysematous cholecystitis was achieved based on survey abdominal radiographs, a barium contrast gastrointestinal series, and abdominal ultrasound. Diagnosis and medical and surgical management of the condition are discussed.

Endothelial cell-specific transcriptional targeting from a hybrid long terminal repeat retrovirus vector containing human prepro-endothelin-1 promoter sequences.

For many applications, specificity of gene expression by recombinant retroviral vectors is necessary. We wished to obtain transcriptional targeting in endothelial cells as part of an antivasculature approach to cancer treatment and have achieved specificity by using the promoter for human prepro-endothelin-1. In particular, we have inserted this heterologous promoter within the 3' long terminal repeat (LTR), replacing all viral upstream transcriptional regulatory sequences, to generate a hybrid LTR with precise fusion at the TATA box for initiation of transcription at the viral start site. Reverse transcription and integration resulted in duplication of this hybrid promoter in the 5' LTR of the provirus for transcription of the internal transgene. An important feature of our vectors is the absence of a selectable marker gene or additional promoters to avoid potential complications of silencing or interference and because selection will be inappropriate for clinical application. This vector design showed endothelial cell specificity of beta-galactosidase expression when tested on a panel of human cell lines and primary breast microvascular endothelial cells, matching the specificity of expression of the endogenous promoter. Such simplified vectors exhibiting transcriptional specificity are likely to be useful for the development of a gene therapy approach to targeting tumor vasculature.

In vivo transfer of bacterial marker genes results in differing levels of gene expression and tumor progression in immunocompetent and immunodeficient mice.

To optimize gene delivery for the treatment of malignant mesothelioma, expression of the beta-galactosidase marker gene was examined in a murine model of intraperitoneal malignant mesothelioma. The beta-galactosidase gene was delivered to the peritoneal cavity of tumor-bearing mice by various plasmid-liposome complexes or by replication-incompetent retrovirus, used alone or complexed to liposomes. In tumor samples from immunodeficient nude mice, moderate levels of gene expression were achieved by liposome-complexed plasmids. Retroviral gene delivery was more effective, and was increased nearly 10-fold by complexing the retrovirus to liposomes. In contrast, in tumor samples from immunocompetent CBA mice treated with the same vectors, no marker gene expression was detected. In immunodeficient mice, tumor growth was not affected by beta-galactosidase gene transfer. However, immunocompetent mice showed a significant decrease in tumor size and increase in survival time after beta-galactosidase delivery. Induction of cytotoxic T cells capable of lysing beta-Gal-transfected tumor cells suggests that tumor cells transduced with the bacterial beta-galactosidase gene may be eliminated in immunocompetent hosts. Our findings also indicate that plasmid-liposome complexes, which achieve a low level of gene expression, and retrovirus-liposome complexes, which result in nearly 100 times higher levels of gene expression in tumor cells in vivo, are similarly effective in inducing an antitumor immune response.

Football injuries.

Players of football will suffer injury. These injuries with their potential effects on the rest of the athlete's lives should be treated with aggressive recognition and rehabilitation of acute injuries. Care should be taken when interpreting published data on football injuries, as the definition of "injury" may widely vary. The most effective treatment of any of the injuries reviewed is prevention. Preseason conditioning, attention to safe technique, and appropriate use of equipment are key preventive strategies. Recently the seriousness of even mild traumatic brain injury (and the second impact syndrome) has reached national consensus. Return to play decisions will be executed most effectively if a clear plan for handling these situations is in place before the injuries take place. Overall, sports medicine research and education have significantly reduced the frequency and seriousness of injuries suffered during football.

Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo.

Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.

Sensitization of rhabdo-, lenti-, and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation.

Vesicular stomatitis virus, human immunodeficiency virus type 2, and human foamy virus, which were produced by cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars, were found to be less stable in human serum than those from alphaGal-negative cells, indicating that galactosyl(alpha1-3)galactosylation sensitizes these viruses as well as mammalian type C oncoviruses (Rother et al., J. Exp. Med. 182:1345-1355, 1995; Takeuchi et al., Nature (London) 379:85-88, 1996) to complement killing via natural anti-alphaGal antibodies. Thus, virus killing mediated by anti-alphaGal antibodies may play a role as a barrier to animal-to-human infection of various enveloped viruses. Virus vectors for human in vivo gene therapy based on the viruses mentioned above should be produced from alphaGal-negative cells.

Comparison of efficiency of infection of human gene therapy target cells via four different retroviral receptors.

The relative efficiency of transduction of gene therapy target cells was measured for retroviruses bearing the envelopes of amphotropic murine leukemia virus (MLV-A), xenotropic murine leukemia virus (MLV-X), gibbon ape leukemia virus (GALV), feline leukemia virus subgroup B (FeLV-B), and the feline endogenous virus RD114. These viruses use various cell-surface receptors. Activated peripheral blood lymphocytes (PBL) and primary melanoma cultures were infected relatively poorly by MLV-X pseudotypes. RD114 pseudotypes infected PBL relatively well, whereas bone marrow progenitor cells were efficiently infected by all viruses. Helper-free virus bearing the envelopes of MLV-A, RD114, or GALV was similarly tested. All infected melanoma or bone marrow progenitor cells efficiently, whereas MLV-A was relatively inefficient for infection of PBL. The general utility of RD114 pseudotyped virus for gene delivery coupled with its resistance to inactivation by human serum makes this envelope the most suitable choice for in vivo gene therapy.

Efficient retroviral transduction of human bone marrow progenitor and long-term culture-initiating cells: partial reconstitution of cells from patients with X-linked chronic granulomatous disease by gp91-phox expression.

The primary immunodeficiencies are attractive candidates for the development of gene therapy approaches based on the transduction of hematopoietic cells. We have constructed a high-titer recombinant retrovirus for expression of gp91-phox, deficiencies of which cause the X-linked form of chronic granulomatous disease (X-CGD). We have used this vector to transduce human bone marrow, using either unfractionated mononuclear cells or purified CD34+ cells as targets and evaluated several infection protocols. Efficient gene transfer to progenitors and long-term culture-initiating cells (LTC-IC) was obtained for each target population. Importantly for potential clinical application, this could be achieved without the use of exogenous cytokines or polybrene. Progenitors representing each of the lineages detectable in vitro were transduced at equal efficiencies. The vector was shown partially to restore gp91-phox deficiency and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in transduced cells derived from X-CGD patients. These data demonstrate that it is possible to transduce primitive human hematopoietic cells efficiently and reconstitute NADPH oxidase.

Detection of gp91-phox precursor protein in B-cell lines from patients with X-linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis.

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.

Sensitization of cells and retroviruses to human serum by (alpha 1-3) galactosyltransferase.

Mammalian C-type retroviruses are inactivated by human serum, following triggering of the classical complement cascade. This may have inhibited transmission to humans of C-type oncoviruses from other mammals. Indeed, the retroviruses human immunodeficiency virus and human T-cell leukaemia virus are resistant to human complement. Antibody-independent activation of human C1q, the first component of the classical pathway, by retroviral envelope proteins has been described. However, retroviruses produced from human cells are resistant to inactivation by human complement and human serum is known to contain antibodies directed against carbohydrates on retroviral envelopes. Gal(alpha 1-3)Gal terminal carbohydrates are expressed by most mammals but are absent in humans, which lack a functional (alpha 1-3)galactosyltransferase gene. Here, we demonstrate that anti-Gal(alpha 1-3)Gal antibodies in human serum inactivate retroviruses produced from animal cells. Expression of porcine (alpha 1-3)galactosyltransferase in human cells renders the cells and the retroviruses they produce sensitive to human serum.

p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox.

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91-phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91-phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.

Generation of interleukin-2-secreting melanoma cell populations from resected metastatic tumors.

This study aimed to determine the feasibility of producing patient-specific, interleukin-2 (IL-2)-secreting tumor cell vaccines for the treatment of metastatic melanoma. Primary tumor cell cultures were established from 26/33 resected metastatic melanoma samples. Recombinant retroviral gene transfer and expression in these cultures was optimized using an amphotropic, defective retrovirus carrying the LacZ gene. All cell cultures were infectable; those that proliferated more rapidly were infected at a higher frequency. Addition of fibroblast growth factor to the culture medium increased the rate of cell proliferation and the efficiency of infection. A single infection with an identical retrovirus carrying a human IL-2 cDNA resulted in the generation of unselected cell populations secreting up to 300 units IL-2/10(6) cells.48 hr. Multiple infections increased the level of IL-2 secretion to 5,000 units/10(6) cells.48 hr. The recombinant viral genome could be detected at approximately single copy in the multiply infected cells; no helper virus was detected. IL-2 secretion from infected cultures was maintained following cryopreservation and x-irradiation. These data demonstrate that heterogeneous tumor cell populations secreting IL-2 can be generated from individual patients to be used as autologous, irradiated cell vaccines.

X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X-linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.