Biomedical subjects

M G Castro

Publications and source records attributed to M G Castro.

At least 37 records · Page 2Linked to original sources

PubMed2002

Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis.

Gene therapy for pituitary disease requires evaluation for safety as well as efficacy. We have reported results of adenovirus-mediated gene transfer using the sheep as a large animal model that allows longitudinal evaluation of hormone secretion and have confirmed high levels of transgene expression up to 7 days after direct stereotaxic injection into the pituitary gland. Here we report the results of detailed histological examination of the pituitary glands from animals injected with two recombinant adenoviruses expressing the beta-galactosidase marker gene, or with saline vehicle to control for the potential tissue-disruptive effect of the injection volume itself. Pituitaries injected with saline showed no evidence of inflammatory response apart from occasional minor foci of apoptosis. In all other respects they were indistinguishable from normal uninjected control pituitary glands. Glands injected with recombinant adenoviruses containing either the hCMV-beta-gal or the hPRL-beta-gal transgene, on the other hand, displayed variable degrees of inflammatory response, with periglandular fibrosis, lymphocytic infiltrate and venulitis in almost all cases. Focal necrosis and/or apoptosis was noted in six of nine cases. In summary, we have found evidence of severe inflammatory reaction within the first seven days of adenovirus injection, amounting to significant hypophysitis. The histological extent of this reaction has not previously been recognised by studies of the efficacy of gene transfer in rodents, and was underestimated by immunocytochemical studies of hormone and transgene expression. The findings emphasise the need for careful evaluation of the safety of endocrine gene therapy, and for caution with the dose of vector used.

Adenoviridae↗

PubMed2001

Adenovirus-mediated gene transfer of a secreted transforming growth factor-beta type II receptor inhibits luminal loss and constrictive remodeling after coronary angioplasty and enhances adventitial collagen deposition.

BACKGROUND: Extracellular matrix (ECM) remodeling is central to the development of restenosis after coronary angioplasty (PTCA). As a regulator of ECM deposition by vascular cells, substantial evidence implicates transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of restenosis. We investigated the effects of intracoronary expression of a transgenic antagonist of TGF-beta1 on luminal loss after PTCA. METHODS AND RESULTS: Porcine coronary arteries were randomized to receive a recombinant adenovirus expressing a secreted form of TGF-beta type II receptor (Ad5-RIIs), an adenovirus expressing beta-galactosidase (Ad5-lacZ), or vehicle only by intramural injection at the site of PTCA. Computerized morphometry 28 days after angioplasty revealed a greater minimum luminal area in Ad5-RIIs-injected arteries (1.71+/-0.12 mm(2)) than in the Ad5-lacZ (1.33+/-0.13 mm(2)) or vehicle-only (1.08+/-0.17 mm(2); P=0.010 by ANOVA) groups. This was accompanied by greater areas within the internal (P=0.013) and external (P=0.031) elastic laminae in Ad5-RIIs-treated vessels. Adventitial collagen content at the site of injury was increased in the Ad5-RIIs group, in contrast to decreases in the Ad5-lacZ and vehicle-only groups (P=0.004). CONCLUSIONS: Adenovirus-mediated antagonism of TGF-beta1 at the site of PTCA reduces luminal loss after PTCA by inhibiting constrictive remodeling. Antagonism of TGF-beta1 stimulates the formation of a dense collagenous adventitia, which prevents constrictive remodeling by acting as an external scaffold. These findings demonstrate the potential of gene therapy-mediated antagonism of TGF-beta1 as prophylactic therapy for restenosis.

Adenoviridae↗

PubMed2001

Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.

The utility of first-generation adenovirus vectors for long-term gene transfer in humans is limited by preexisting antiadenoviral immunity. We demonstrate here that new-generation high-capacity adenovirus vectors (HC-Ads) can efficiently transduce the brain and mediate stable transgene expression for at least 2 months, even in the presence of a preexisting antiadenoviral immune response. First-generation vector-mediated transduction was almost completely abolished in preimmunized animals within 60 days of the vector injection. Levels of HC-Ad-mediated transduction by 3 days postinjection were not significantly affected by preimmunization, were reduced within 14 days to 56% of those levels seen in nonimmunized animals, and remained stable until day 60 postinjection. Acute brain inflammation elicited by the HC-Ad vector injection was more transient, and was reduced in intensity compared with brain inflammation elicited by the first-generation vector injection in immunized animals. Inflammation was significantly higher in all immunized animals than in nonimmunized animals. Our results show that preexisting antiadenoviral immunity does not significantly reduce initial HC-Ad-mediated infection of the brain and is not a barrier to stable HC-Ad vector-mediated transduction of the CNS. Although input HC-Ad capsid proteins injected into the brain may contain transient targets for a brain-infiltrating cellular adenovirus-specific immune response, this fails to eliminate transgene expression. Thus HC-Ads show promise for gene therapy of chronic brain disease.

Adenoviridae↗

PubMed2001

Acute direct adenoviral vector cytotoxicity and chronic, but not acute, inflammatory responses correlate with decreased vector-mediated transgene expression in the brain.

The potential utility of adenoviruses for the treatment of chronic neurological disease is controversial due to reports of vector-associated toxicity, inflammation, and transient transgene expression. To focus upon the mechanism by which transgene expression is lost, we injected increasing doses [1 x 10(6) to 1 x 10(9) infectious units (iu)] of a first-generation adenovirus vector expressing beta-galactosidase into the brains of immune-competent adult rats. Transgene expression was evaluated simultaneously with acute neuronal and glial cell cytotoxicity, and acute and chronic inflammation using immunohistochemistry, at 3 and 30 days post-vector administration. Our results show a clear threshold effect of viral dose upon the amount of transgene expression persisting by 30 days after vector administration. Below 10(8) iu, transgene expression remained stable over the 30-day period. Following infection of more than 10(8) iu, the extent of transgene expression at 30 days was inversely correlated with increasing viral dose. The severity of acute inflammation increased proportionally with increasing vector dose from 10(6) to 10(9) infectious units. In contrast, acute vector-mediated cytotoxicity and chronic inflammation were observed only above the threshold level of vector dose. Above 10(8) iu both the extent of the acute toxicity and the severity of the chronic inflammation were inversely correlated with transgene expression at 30 days. Thus, our data suggest that both an acute loss of cells through direct vector-mediated toxicity and the elicitation of chronic inflammation (but not acute inflammation) may account for the decline in transduction persistence at high vector doses.

Adenoviridae↗

PubMed2001

Towards global and long-term neurological gene therapy: unexpected transgene dependent, high-level, and widespread distribution of HSV-1 thymidine kinase throughout the CNS.

One of the challenges of neurological gene therapy for the treatment of chronic neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, is achieving high levels, widespread distribution, and long-lived transgene expression in the brain. Here, following the intracerebral injection of a recombinant adenovirus (RAd) encoding herpes simplex virus type 1 thymidine kinase (HSV1-TK), we detect very high levels of HSV1-TK immunoreactivity throughout the brain both ipsilaterally and contralaterally to the injection site, for up to 12 months following vector administration. This study concludes that long-term, high-level, and anatomically distributed HSV1-TK immunoreactivity can be obtained, and that this is most likely due to transgene-specific properties, because neither the distribution nor the longevity were observed for the transgene beta-galactosidase encoded by a co-injected vector. Thus, we demonstrate that transgene expression can be achieved over widespread areas of the rodent brain, even 12 months after a single injection of first-generation adenovirus vector.

Adenoviridae↗

PubMed2001

Regulated, adenovirus-mediated delivery of tyrosine hydroxylase suppresses growth of estrogen-induced pituitary prolactinomas.

Prolactin-secreting adenomas are one of the most common types of intracranial neoplasm found in humans. The modalities of clinical treatment currently in use include D(2)-dopamine receptor agonists, surgery, and radiotherapy, and the success rates for treatment are good. However, there are prolactinomas that are difficult to treat. As an alternative, we have developed a gene therapy strategy in which the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is overexpressed in the anterior pituitary (AP) gland. Because dopamine is known to have an inhibitory effect on lactotroph growth and prolactin secretion, we developed a system that would enable its local synthesis from freely available precursor amino acids. A dual adenovirus tetracycline-regulatable expression system was generated to control the production of TH. In the absence but not presence of the tetracycline analog doxycycline, TH expression was observed in AP tumor cell lines AtT20, GH3, and MMQ. In both primary AP cell cultures and the AP gland, in situ expression of TH was seen in lactotrophs, somatotrophs, corticotrophs, thyrotrophs, and gonadotrophs in the absence but not presence of doxycycline. The ability of this system to inhibit hyperprolactinemia and pituitary lactotroph hyperplasia was then assessed in a model of estrogen- or estrogen/sulpiride-induced pituitary tumors. In the absence but not presence of doxycycline, a 49% reduction in pituitary growth and 58% reduction in the increase of circulating prolactin levels were observed in estrogen, but not estrogen/sulpiride, treated rats. These results indicate that in situ dopamine enhancement gene therapy can be a useful tool for the treatment of prolactinoma. Dopamine synthesis can be tightly regulated and the therapeutic benefit of the system is only inhibited when local dopamine signaling is impaired.

Adenoviridae↗

PubMed2001

Genetic engineering within the adult brain: implications for molecular approaches to behavioral neuroscience.

Currently, the most popular technology used to modify the molecular makeup of the nervous system is through germline modifications of early embryos. This allows to construct gene 'knock-ins' (gene overexpression) or 'knock-outs' (gene deletions). This technology leads to gene additions or deletions from the earliest developmental stages. This can potentially lead to compensatory genetic changes. The technology to achieve inducible and cell-type-specific changes in gene expression in transgenic animals has been established. However, it is not yet possible, to reliably turn a particular gene 'on' or 'off' exclusively in adult animals. Alternatively, the use of gene transfer technology in fully mature animals could overcome many of these shortcomings. Gene therapy is the use of nucleic acids as drugs, and uses gene transfer technology to genetically engineer adult animals. Viral and nonviral vectors have been modified to serve as vectors for nucleic acid sequences of interest. Thus, over the last two decades, methods have been developed to deliver particular nucleic acids directly to target tissues. Further technological advances allow delivery of transgenes or antisense mRNAs directly to predetermined cell types, as well as their delivery under the control of inducible promoter elements. Combined transgenic (i.e., germline modifications) and viral vector technology will also be very powerful in allowing the genetic modification of selected neuronal populations in adult animals. In this review, we discuss the potential of gene delivery to the brain to analyze the effect of genetic engineering of particular neuronal groups on behavior, as well as recent developments and applications of newly engineered vector systems to allow transgenesis within nervous structures of adult animals.

Animals↗

PubMed2001

Molecular therapy in a model neuroendocrine disease: developing clinical gene therapy for pituitary tumours.

The main objectives of pituitary tumour treatment are to restore normal function of the pituitary gland and prevent tumour recurrences. In spite of the success of current therapies in the treatment of relatively small tumours, new therapeutic alternatives need to be explored for large invasive tumours, tumour recurrences postsurgery, and when intolerance to drug treatment develops. Gene therapy, which uses nucleic acids as drugs, is a very attractive alternative to classic therapeutic modalities. With the development of efficient gene delivery vectors, which allow widespread distribution and long-term transgene expression with limited side effects, the clinical implementation of gene therapy for the treatment of pituitary tumours will become a reality within the next five to ten years.

Adenoviridae↗

PubMed2001

Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination.

Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus. By using a very efficient recombinase, in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography-based virus purification.

Adenoviridae↗

PubMed2001

Adenovirus-mediated expression of HSV1-TK or Fas ligand induces cell death in primary human glioma-derived cell cultures that are resistant to the chemotherapeutic agent CCNU.

Due to minimal treatment success with surgery, radiotherapy, and chemotherapy, the aim of this study was to test the therapeutic potential of gene therapy for the treatment of glioblastoma multiforme (GBM). We have quantitatively analyzed two gene therapy approaches using short-term human glioma cell cultures derived from surgical biopsies (designated IN859, IN1612, IN2045, IN1760, and IN1265) and compared the results of gene therapy with the chemosensitivity of the same cells. All of the glioma cell cultures tested were susceptible to recombinant adenovirus (RAd)-mediated infection. Expression of herpes simplex virus type 1-thymidine kinase (RAd128), followed by ganciclovir treatment, induced apoptosis in all of the glioma cell cultures studied, including three that are resistant to the chemotherapeutic drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). Expression of murine Fas ligand (RAdhCMV-mFasL) also induced cell death in four of the five cell cultures studied. One cell culture that was resistant to CCNU was also resistant to apoptosis induced by mFasL expression. These results suggest that sensitivity to chemotherapeutic agents does not necessarily correlate with the sensitivity to gene therapy treatments. RAds expressing therapeutic gene products in human glioma cell cultures are able to induce apoptosis even in some cells that are resistant to a commonly used chemotherapeutic agent. Therefore, RAd-mediated gene transfer could be a good candidate to further develop gene therapy for the treatment of GBM.

Adenoviridae↗

PubMed2001

Death receptor-independent cytochrome c release and caspase activation mediate thymidine kinase plus ganciclovir-mediated cytotoxicity in LN-18 and LN-229 human malignant glioma cells.

Suicide gene therapy using viral transfer of herpes simplex virus type I (HSV-1) thymidine kinase (TK) and subsequent ganciclovir (GCV) chemotherapy was the first approach used in clinical trials of somatic gene therapy for glioblastoma. The molecular pathways mediating TK/GCV-induced cell death remain to be elucidated. Here, we report that adenoviral (Ad)-TK/GCV-induced death is p53-independent and does not involve altered CD95 or CD95L expression. Ectopic expression of the preferential caspase 8 inhibitor, crm-A, inhibits Ad-CD95L-induced cell death but has no effect on TK/GCV cytotoxicity. LN-18 glioma cells selected for resistance to death receptor-mediated cell death do not acquire cross-resistance to TK/GCV. TK/GCV triggers mitochondrial cytochrome c release and activation of caspases 3, 7, 8 and 9 in a death receptor-independent manner. These events are associated with the loss of BCL-X(L). Forced expression of a BCL-X(L) transgene, or co-exposure to a pseudosubstrate caspase inhibitor, zVAD-fmk, inhibit TK/GCV cytotoxicity. Double-transfected cell lines expressing crm-A and enhanced green fluorescent protein (eGFP) show that the bystander effect in vitro is also death receptor- and caspase 8-independent. TK/GCV therapy does not kill glioma cells in synergy with cancer chemotherapy drugs, including lomustine, temozolomide and topotecan. In contrast, there is strong synergy of TK/GCV and CD95L. Thus, TK/GCV-induced cell death involves a mitochondria-dependent loop of caspase acvtivation that can be synergistically enhanced by death receptor agonists such as CD95L. TK/GCV-mediated sensitization of glioma cells to CD95L expressed on immune effector cells or parenchymal brain cells might account for the immune system's and bystander effects of TK/GCV therapy observed in rodent glioma models in vivo.

Adenoviridae↗

PubMed2001

Photoperiodic regulation of prolactin gene expression in the Syrian hamster by a pars tuberalis-derived factor.

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8-12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.

Animals↗

PubMed2001

Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells.

Pertussis toxin (PT) comprises an active subunit (S1), which ADP-ribosylates the alpha subunit of several mammalian G proteins, and the B oligomer (S2-S5), which binds glycoconjugate receptors on cells. In a previous report, expression of S1 in Cos cells resulted in no observable cytotoxicity, and it was hypothesized that either S1 failed to locate its target proteins or the B oligomer was also necessary for cytotoxicity. To address this, we stably transfected S1 with and without a signal peptide into mammalian cells. Immunofluorescence analysis confirmed the function of the signal peptide. Surprisingly, we found that S1 was active in both transfectants, as determined by clustering of transfected Chinese hamster ovary (CHO) cells and ADP-ribosylation of G proteins. Constructs with a cysteine-to-serine change at residue 201 or a truncated S1 (residues 1-181) were also active when transfected into cells. Constructs with an inactive mutant S1 had no activity, confirming that the observed results were due to the activity of the toxin subunit. We conclude that S1 is active when expressed in mammalian cells without the B oligomer, that secretion into the endoplasmic reticulum does not prevent this activity and that the C-terminal portion of S1 is not required for its activity in cells.

Adenosine Diphosphate Ribose↗

PubMed2001

Long-term transgene expression within the anterior pituitary gland in situ: impact on circulating hormone levels, cellular and antibody-mediated immune responses.

Adenoviral vectors have been identified as useful tools for gene transfer to the pituitary gland with the aim of providing therapeutic treatments for pituitary diseases. Although successful adenovirus-mediated gene transfer to the pituitary has been shown, the duration of transgene expression, local immune responses and consequences on circulating pituitary hormone levels have not been investigated. These are critical not only for the successful implementation of these gene transfer techniques both for physiological and/or therapeutic applications but also for assessing the safety of these approaches. We have therefore assessed duration and levels of transgene expression 3 days, 14 days, 1, 2, and 3 months after delivery of adenoviruses expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK), under the control of the major immediate early human cytomegalovirus (RAd-hCMV/TK) or human PRL (RAd-hPrl/TK) promoters, to the anterior pituitary (AP) gland in situ. The presence of vector genome and cellular immune infiltrates within the AP gland were also studied along with the levels of circulating anti-adenovirus neutralizing antibodies and AP hormones in sera. Ubiquitous or cell-type specific expression of HSV1-TK within the AP gland was seen from RAd-hCMV/TK and RAd-hPrl/TK respectively at all time points, although a reduction in expression was seen over time. PCR amplification of HSV1-TK specific sequences showed the persistence of adenoviral genomes for up to 3 months. Analysis of the AP showed the presence of a virus-induced inflammation that peaked around day 14 and was resolved between 2-3 months. ED1-positive macrophages, CD8-positive T-cells and CD161-positive NK cells were identified up to 1 month after virus administration. A virus-induced humoral immune response was also present as anti-adenovirus neutralizing antibodies were detected from 14 days after virus administration. Levels of circulating pituitary hormones were unaffected by virus administration with the exception of the stress hormone ACTH which was increased at 3 days but normalized by 14 days. In conclusion, our data indicates that adenovirus-mediated delivery to the AP gland in situ may be a useful tool for the treatment of pituitary diseases as no major cytotoxicity or disruption of AP hormonal functions are seen. Despite of this, further developments to this approach still need to be made to combat the reduced transgene expression seen over time and the induction of virus-induced immune responses.

Adenoviridae↗

PubMed2001

Cell type-specific adenoviral transgene expression in the intact ovine pituitary gland after stereotaxic delivery: an in vivo system for long-term multiple parameter evaluation of human pituitary gene therapy.

Ablative therapies for pituitary tumors commonly cause irreversible damage to normal pituitary cells. Toxin gene therapy should therefore ideally be targeted to specific cell types to avoid collateral cell damage. To evaluate cell-type-specific adenoviral gene transfer in the intact pituitary gland we have used stereotaxic transcranial delivery of recombinant adenoviruses in the sheep with continuous assessment of endocrine function. Adenoviral ss-galactosidase expression was driven either by the human cytomegalovirus (hCMV) promoter or the human PRL gene promoter. The hCMV promoter directed adenoviral ss-galactosidase expression in all pituitary cell types, but the PRL promoter restricted this exclusively to lactotropic cells, indicating that this promoter conferred appropriate cell type specificity in the context of adenoviral transduction in vivo. Serial measurements of plasma hormones showed no disruption of endocrine function over 7 days after intrapituitary injection. In summary, this work shows cell type-specific expression of an adenoviral transgene in the mixed cell population of the intact pituitary gland in vivo in a large animal model and indicates that stereotaxic intrapituitary delivery does not disrupt normal endocrine function.

Adenoviridae↗

PubMed2001

Switching on and off transgene expression within lactotrophic cells in the anterior pituitary gland in vivo.

To further develop our understanding of anterior pituitary (AP) function and to aid the development of gene therapy strategies for the treatment of pituitary diseases, adenovirus (Ad)-mediated gene transfer to the AP gland will be a useful tool. Although successful widespread gene transfer within the AP has been achieved using first generation Ads the ability to control transgene expression would be very beneficial when studying AP regulatory functions and delivering a potentially therapeutic gene into the AP gland. A dual adenoviral vector system encoding for cell type-specific and regulatable transcription units was developed to achieve transcriptionally targeted transgenesis within specific cell populations in the adult AP gland. To achieve regulatable transgene expression within predetermined AP cells, the tetracycline-responsive transcriptional elements have been engineered to be under the control of human, lactotroph-specific PRL (hPRL) promoter elements within a dual adenoviral vector system. The inducibility, cell type specificity, and levels of transgene expression were characterized in vitro and in vivo and compared with the strong ubiquitous beta-actin/human cytomegalovirus (CAG) promoter. Inducible expression of the marker gene beta-galactosidase under the control of the hPRL promoter was restricted to lactotrophic tumor cell lines and lactotrophic cells within primary AP cultures. Lactotroph cell type specificity and inducible transgene expression were also observed within the AP gland in vivo, and this could be switched on or off. Administration of doxycycline abrogated transgene expression both in vitro and in vivo. Our results also provide evidence that an excess of trans-activator is needed to achieve maximal transgene expression. Our data indicate that combined transcriptional and inducible transgenesis can be achieved using adenoviral vectors that allow spatial and temporal restriction of transgene expression within the adult AP gland in vivo.

Adenoviridae↗

PubMed2001

Gene therapy for Parkinson's disease: recent achievements and remaining challenges.

Gene therapy is the use of nucleic acids as drugs. Thus, ways had to be developed to deliver this new generation of drugs to target tissues. Various viral and non-viral vectors have been engineered to carry potentially therapeutic nucleic acids into diseased organs or target cells. The brain offers a particular challenge for gene delivery to its constituent cells: it is encased by the skull, separated from the general circulation by the blood brain barrier, and made up of mostly non-dividing cells. The skull limits direct injection of vectors into the brain, the blood brain barrier inhibits the easy entry of vectors injected into the bloodstream, and post mitotic target cells restrict what type of vector can be used to deliver genes to the brain. We will discuss the main challenges faced by gene delivery to the brain, i.e. immune responses to the delivery vectors and therapeutic transgenes and length of duration of the therapy specifically as applied to Parkinson's disease. We will also discuss therapeutic strategies, which could be implemented to treat Parkinson's disease, and the models in which they have been tested.

Animals↗

PubMed2000

Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases.

Although adenoviral vectors provide prolonged gene expression in the brain by comparison to peripheral organs, expression is eliminated by a severe inflammatory infiltration (i.e., activated macrophages/microglia and T-lymphocytes) after peripheral infection with adenovirus. Here, we demonstrate that high-capacity adenoviral (HC-Ad) vectors succeed in maintaining long-term transgene expression in the brain, even in the presence of an active peripheral immunization with adenovirus that completely eliminates expression from first-generation vectors within 60 days. Importantly, even 60 days after the peripheral infection, brains injected with first-generation vectors exhibited evidence of a chronic infiltration of CD8(+) cells, macrophage/microglial activation, and up-regulation of brain MHC-I expression. No inflammation was observed in the brains injected with the HC-Ad vector. Thus, these results demonstrate that HC-Ad vectors will allow safe, stable, and long-term transgene expression in the brain, even in the presence of peripheral infection with adenovirus. This markedly improves the prospects for the use of adenoviral vectors for long-term gene therapy of neurological disorders.

Adenoviridae Infections↗