Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters.

Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.

Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy.

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

siRNA silencing of keratinocyte-specific GFP expression in a transgenic mouse skin model.

Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and 'patient-friendly' siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.

Short-duration-focused ultrasound stimulation of Hsp70 expression in vivo.

The development of transgenic reporter mice and advances in in vivo optical imaging have created unique opportunities to assess and analyze biological responses to thermal therapy directly in living tissues. Reporter mice incorporating the regulatory regions from the genes encoding the 70 kDa heat-shock proteins (Hsp70) and firefly luciferase (luc) as reporter genes can be used to non-invasively reveal gene activation in living tissues in response to thermal stress. High-intensity-focused ultrasound (HIFU) can deliver measured doses of acoustic energy to highly localized regions of tissue at intensities that are sufficient to stimulate Hsp70 expression. We report activation of Hsp70-luc expression using 1 s duration HIFU heating to stimulate gene expression in the skin of the transgenic reporter mouse. Hsp70 expression was tracked for 96 h following the application of 1.5 MHz continuous-wave ultrasound with spatial peak intensities ranging from 53 W cm(-2) up to 352 W cm(-2). The results indicated that peak Hsp70 expression is observed 6-48 h post-heating, with significant activity remaining at 96 h. Exposure durations were simulated using a finite-element model, and the predicted temperatures were found to be consistent with the observed Hsp70 expression patterns. Histological evaluation revealed that the thermal damage starts at the stratum corneum and extends deeper with increasing intensity. These results indicated that short-duration HIFU may be useful for inducing heat-shock expression, and that the period between treatments needs to be greater than 96 h due to the protective properties of Hsp70.

Integrating the biological characteristics of oncolytic viruses and immune cells can optimize therapeutic benefits of cell-based delivery.

Despite significant advances in the development of tumor-selective agents, strategies for effective delivery of these agents across biological barriers to cells within the tumor microenvironment has been limiting. One tactical approach to overcoming biological barriers is to use cells as delivery vehicles, and a variety of different cell types have been investigated with a range of agents. In addition to transporting agents with targeted delivery, cells can also produce their own tumoricidal effect, conceal a payload from an immune response, amplify a selective agent at the target site and facilitate an antitumor immune response. We have reported a therapeutic combination consisting of cytokine induced killer cells and an oncolytic vaccinia virus with many of these features that led to therapeutic synergy in animal models of human cancer. The synergy was due to the interaction of the two agents to enhance the antitumor benefits of each individual component. As both of these agents display broad tumor-targeting potential and possess unique tumor killing mechanisms, together they were able to recognize and destroy a far greater number of malignant cells within the heterogeneous tumor than either agent alone. Effective cancer therapy will require recognition and elimination of the root of the disease, the cancer stem cell, and the combination of CIK cells and oncolytic vaccinia viruses has this potential. To create effective tumor-selective agents the viruses are modified to take advantage of the unique biology of the cancer cell. Similarly, if we are to develop targeted therapies that are sufficiently multifaceted to eliminate cancer cells at all stages of disease, we should integrate the virus into the unique biology of the cell delivery vehicle.

Combining immune cell and viral therapy for the treatment of cancer.

A variety of viral-based and immune cell therapies have been proposed for use in the treatment of cancer. One possible approach to improve the effectiveness of these biological agents may be to combine them such that we can take advantage of natural immune cell-pathogen relationships. Here we discuss these potential approaches with particular emphasis on the use of immune cells as carrier vehicles to deliver viral therapies to the tumor.

Cellular tolerance to pulsed hyperthermia.

Transient heating of tissues leading to cellular stress or death is very common in medicine and biology. In procedures involving a mild (below 70 degrees C) and prolonged (minutes) heating, such as hyperthermal tumor therapy, the cellular response to thermal stress is relatively well studied. However, there is practically no data on cell viability at higher temperatures and shorter exposures, while the demand for this knowledge is growing. Two main reasons motivate this research: (i) a growing number of laser therapies and surgical procedures involving pulsed heating, and (ii) cellular viability data at short exposures to high temperatures provide a unique insight into the understanding of processes leading to thermally induced cellular death. We designed a technique and performed a study of cell viability under pulses of heat from 0.3 to 100 ms in duration with peak temperatures as high as 130 degrees C. We found that the threshold of cellular death in this range can be accurately approximated by the Arrhenius law with the activation energy of 1 eV, a significantly lower value than was reported in studies based on multisecond exposures.

Advancing animal models of neoplasia through in vivo bioluminescence imaging.

Malignant disease is the final manifestation of complex molecular and cellular events leading to uncontrolled cellular proliferation and eventually tissue destruction and metastases. While the in vitro examination of cultured tumour cells permits the molecular dissection of early pathways in tumorigenesis on cellular and subcellular levels, only interrogation of these processes within the complexity of organ systems of the living animal can reveal the full range of pathophysiological changes that occur in neoplastic disease. Such analyses require technologies that facilitate the study of biological processes in vivo, and several approaches have been developed over the last few years. These strategies, in the nascent field of in vivo molecular and cellular imaging, combine molecular biology with imaging modalities as a means to real-time acquisition of functional information about disease processes in living systems. In this review, we will summarise recent developments in in vivo bioluminescence imaging (BLI) and discuss the potential of this imaging strategy for the future of cancer research.

Bioluminescence imaging of lymphocyte trafficking in vivo.

Lymphocytes are highly mobile cells that travel throughout the body in response to a tremendous variety of stimuli. Revealing lymphocyte trafficking patterns in vivo is necessary for a complete understanding of immune function, as well as cell-cell and cell-tissue interactions in immune development and in response to insult. Although the location of cell populations in various tissues at any given point in time may be revealed by techniques such as flow cytometry and immunofluorescence, these methods are not readily amenable to the assessment of dynamic cell migration patterns in vivo. In the past 5 years, technologies for imaging molecular and cellular changes in living animals have advanced to a point where it is possible to reveal the migratory paths of these vitally important cells. Here, we review one advancement in cellular imaging, in vivo bioluminescence imaging, which addresses the problem of lymphocyte tracking. This imaging strategy has the potential to elucidate the temporal patterns of immune responses and the spatial distribution of lymphocytes within the body.

Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression.

The use of transgenic animals in biomedical research is increasing rapidly and may be the best means of determining gene function. Generating transgenic animals typically requires time-consuming screening processes, and gene function is assessed by an array of difficult phenotypic and biochemical assays performed ex vivo. To address the unmet need in transgenic research for functional assays performed with ease in living animals, we demonstrate here that in vivo detection of luciferase enzyme as a transcriptional reporter facilitates rapid screening for both the presence and function of transgenes in intact living mice. Using this approach we identified three bioluminescent transgenic founders where the transgene consisted of the heme oxygenase promoter fused to the modified coding sequence of the luciferase gene. These founders were identified from 183 pups and confirmed by PCR analysis. Identification of HO-1-luc homozygotes from back- crossed F2 littermates was then accelerated by in vivo imaging. In another transgenic mouse line, where the transgene was comprised of the bone morphogenic-4 (BMP4) promoter fused to the modified luciferase gene, we were able to identify transgenic animals and in each line we were able to visualize patterns of expression in living animals over time. The light production from these transgenic mice indicated that the desired DNA fragment was functional and different expression profiles apparent at different ages and after gene induction.

Adoptive immunotherapy of experimental autoimmune encephalomyelitis via T cell delivery of the IL-12 p40 subunit.

CD4+ T cells are believed to play a central role in the initiation and perpetuation of autoimmune diseases such as multiple sclerosis. In the murine model for multiple sclerosis, experimental autoimmune encephalomyelitis, pathogenic T cells exhibit a Th1-like phenotype characterized by heightened expression of proinflammatory cytokines. Systemic administration of "regulatory" cytokines, which serve to counter Th1 effects, has been shown to ameliorate autoimmune responses. However, the inherent problems of nonspecific toxicity limit the usefulness of systemic cytokine delivery as a potential therapy. Therefore, we used the site-specific trafficking properties of autoantigen-reactive CD4+ T cells to develop an adoptive immunotherapy protocol that provided local delivery of a Th1 cytokine antagonist, the p40 subunit of IL-12. In vitro analysis demonstrated that IL-12 p40 suppressed IFN-gamma production in developing and effector Th1 populations, indicating its potential to modulate Th1-promoted inflammation. We have previously demonstrated that transduction of myelin basic protein-specific CD4+ T cells with pGC retroviral vectors can result in efficient and stable transgene expression. Therefore, we adoptively transferred myelin basic protein-specific CD4+ T cells transduced to express IL-12 p40 into mice immunized to develop experimental autoimmune encephalomyelitis and demonstrated a significant reduction in clinical disease. In vivo tracking of bioluminescent lymphocytes, transduced to express luciferase, using low-light imaging cameras demonstrated that transduced CD4+ T cells trafficked to the central nervous system, where histological analysis confirmed long-term transgene expression. These studies have demonstrated that retrovirally transduced autoantigen-specific CD4+ T cells inhibited inflammation and promoted immunotherapy of autoimmune disorders.

Carbon monoxide and bilirubin production in neonates.

Neonatal hyperbilirubinemia is a normal postnatal phenomenon resulting from a transitional imbalance between the production and elimination of bilirubin in the neonate. Bilirubin has been shown to be not only a potent antioxidant, but also toxic at excessive concentrations. As a result, the biology of bilirubin, its production, regulation, and measurements have been the focus of extensive studies. Bilirubin, carbon monoxide, and iron are derived from the degradation of heme, a ubiquitous two-step pathway catalyzed by the enzyme, heme oxygenase. It has been shown that these metabolically active products from the heme catabolic pathway may, in turn, influence many other biologic processes. This report provides a brief overview of these interrelationships in the hope that it may provide insight into the central role this pathway plays in the existence of most organisms.

Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis.

Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen-specific (CII-specific) CD4(+) T hybridomas or primary CD4(+) T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4(+) T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA.

In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression.

Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors that produce long-term expression without toxicity. To investigate future approaches for in utero gene delivery, the efficacy and safety of prenatal administration of rAAV were determined. Using luciferase as a reporter, expression was assessed by whole-body imaging and by analysis of luciferase activity in tissue extracts, at the time of birth and monthly thereafter. Transgene expression was detected in all injected animals. Highest levels of luciferase activity were detected at birth in the peritoneum and liver, while the heart, brain, and lung demonstrated low-level expression. In vivo luciferase imaging revealed persistent peritoneal expression for 18 months after in utero injection and provided a sensitive whole-body assay, useful in identifying tissues for subsequent analyses. There was no detectable hepatocellular injury. Antibodies that reacted with either luciferase or rAAV were not found. AAV sequences were not detected in germ-line tissues of injected animals or in tissues of their progeny. In utero AAV-mediated gene transfer in this animal model demonstrates that novel therapeutic vectors and strategies can be rapidly tested in vivo and that rAAV may be developed to ameliorate genetic diseases with perinatal morbidity and mortality.

In vivo patterns of heme oxygenase-1 transcription.

Gene fusions composed of specific promoters and bioluminescent reporter genes can be used to assess gene expression patterns using whole-body imaging in living animal models. A transgenic mouse model was developed using the regulatory elements of the heme oxygenase promoter to drive luciferase as the reporter gene. In these transgenic mice, heme oxygenase (HO)-1 expression was apparent in neuronal tissues of neonates but not adults as measured by whole-body imaging, and in adults transcription of the reporter gene was inducible by known inducers of HO-1 transcription. Whole-body imaging of luciferase activity was then used to evaluate the effects of metalloporphyrins (Mps) on the transcription of the reporter gene. Some of the Mps, which are potent inhibitors of HO activity, did not activate the reporter gene above background. These Mps are ideally suited as chemotherapeutics that may target bilirubin production rates by inhibiting HO activity, but not result in a net increase in output from the HO gene. In contrast, known inducers of HO transcription did increase luciferase activity as did some of the other Mps that have been examined. Using whole-body in vivo transcriptional assays may facilitate rapid screening of potential therapeutic compounds for both desired and untoward effects.

Genetic analysis of viral variants selected in transmission of human immunodeficiency viruses to newborns.

Our previous studies have indicated that HIV transmission from infected mothers to infants occurs with viruses showing rapid kinetics of replication, and either resistance to maternal neutralizing antibodies or sensitivity to enhancing antibodies. The genotypic patterns that result in these and other phenotypic viral characteristics may provide clues to the selection pressures exerted during this mode of transmission. For this reason, DNA sequences of the envelope gene (env) were determined for viral isolates obtained from seropositive women who were mothers of either infected or uninfected infants. Sequences of viruses isolated early in life from the infected newborns were also determined, such that diversity both within isolates and between maternal and infant isolates could be assessed. Among isolates obtained from mothers of uninfected infants, the V3 region of env demonstrated a higher degree of heterogeneity than those from mothers of infected infants. Similar to the viruses obtained from the mothers of infected infants, the infant-derived viral sequences were relatively homogeneous. Finally, the reactivity of maternal plasma with infant-derived HIV isolates, whether via neutralizing or enhancing antibodies, appeared to predict the distribution of viral sequences in the infant isolates. These data suggest that selective pressure on HIV-1 during transmission or growth in the infected infant may be mediated by biologic and/or immunologic processes.

Use of reporter genes for optical measurements of neoplastic disease in vivo.

Revealing the cellular and molecular changes associated with cancer, as they occur in intact living animal models of human neoplastic disease, holds tremendous potential for understanding disease mechanisms and elucidating effective therapies. Since light is transmitted through mammalian tissues, at a low level, optical signatures conferred on tumor cells by expression of reporter genes encoding bioluminescent and fluorescent proteins can be detected externally using sensitive photon detection systems. Expression of reporter genes, such as the bioluminescent enzyme firefly luciferase (Luc) or variants of green fluorescent protein (GFP) in transformed cells, can effectively be used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies have been evaluated non-invasively in living experimental animals using these reporter genes. Detection of Luc-labeled cells in vivo was extremely sensitive with signals over background from as few as 1000 human tumor cells distributed throughout the peritoneal cavity of a mouse with linear relationships between cell number and signal intensity over five logs. GFP offers the strength of high-resolution ex vivo analyses following in vivo localization of the tumor. The dynamic range of Luc detection allows the full disease course to be monitored since disease progression from small numbers of cells to extensive disease can be assessed. As such, therapies that target minimal disease as well as those designed for late stage disease can be readily evaluated in animal models. Real time spatiotemporal analyses of tumor cell growth can reveal the dynamics of neoplastic disease, and facilitate rapid optimization of effective treatment regimens. Thus, these methods improve the predictability of animal models of human disease as study groups can be followed over time, and can accelerate the development of therapeutic strategies.

Carbon monoxide detection and biological investigations.

Even though the heme degradation pathway consists of only two reactions, it and its major enzyme (i.e. HO), nonetheless, impact other processes not only through the removal of excess heme, but also through the production of several metabolically active compounds. Thus CO and biliverdin along with reactive iron, Fe2, are the primordial products of this ancient, highly conserved reaction. That every component of the heme catabolic pathway is directly or indirectly related to other reactions involving oxygen or light is, perhaps, no accident of nature. That a fundamentally destructive event can be linked with a multiplicity of synthetic events and various biological effects, depending on the timing and location of the HO activity, is testament to the economy and the ultimate beauty of nature. Furthermore, the interaction of the heme catabolic pathway with that of the NOS system may lead to even more exciting avenues of research. It may be shown that the integrity of the heme catabolic pathway, which is ever present and plays a role in every tissue, is central to the existence of most complex organisms.