Nanobodies as probes to investigate purinergic signaling.

Nanobodies (VHHs) are the single variable immunoglobulin domains of heavy chain antibodies (hcAbs) that naturally occur in alpacas and other camelids. The two variable domains of conventional antibodies typically interact via a hydrophobic interface. In contrast, the corresponding surface area of nanobodies is hydrophilic, rendering these single immunoglobulin domains highly soluble, robust to harsh environments, and exceptionally easy to format into bispecific reagents. In homage to Geoffrey Burnstock, the pioneer of purinergic signaling, we provide a brief history of nanobody-mediated modulation of purinergic signaling, using our nanobodies targeting P2X7 and the NAD+-metabolizing ecto-enzymes CD38 and ARTC2.2 as examples.

Potential sealing and repair of human FM defects after trauma with peptide amphiphiles and Cx43 antisense.

OBJECTIVE: We examined whether peptide amphiphiles functionalised with adhesive, migratory or regenerative sequences could be combined with amniotic fluid (AF) to form plugs that repair fetal membrane (FM) defects after trauma and co-culture with connexin 43 (Cx43) antisense. METHODS: We assessed interactions between peptide amphiphiles and AF and examined the plugs in FM defects after trauma and co-culture with the Cx43antisense. RESULTS: Confocal microscopy confirmed directed self-assembly of peptide amphiphiles with AF to form a plug within minutes, with good mechanical properties. SEM of the plug revealed a multi-layered, nanofibrous network that sealed the FM defect after trauma. Co-culture of the FM defect with Cx43 antisense and plug increased collagen levels but reduced GAG. Culture of the FM defect with peptide amphiphiles incorporating regenerative sequences for 5 days, increased F-actin and nuclear cell contraction, migration and polarization of collagen fibers across the FM defect when compared to control specimens with minimal repair. CONCLUSIONS: Whilst the nanoarchitecture revealed promising conditions to seal iatrogenic FM defects, the peptide amphiphiles need to be designed to maximize repair mechanisms and promote structural compliance with high mechanical tolerance that maintains tissue remodeling with Cx43 antisense for future treatment.

Use of vivo-morpholinos for control of protein expression in the adult rat brain.

Vivo-morpholinos are commercially available morpholino oligomers with a terminal octa-guanidinium dendrimer for enhanced cell-permeability. Existing evidence from systemically delivered vivo-morpholinos indicate that genetic suppression can last from days to weeks without evidence of cellular toxicity. However, intravenously delivered vivo-morpholinos are ineffective at protein suppression in the brain, and no evidence is available regarding whether intracranially delivered vivo-morpholinos effectively reduce target protein levels, or do so without inducing neurotoxicity. Here we report examples in which in vivo microinjection of antisense vivo-morpholinos directed against three different targets (xCT, GLT1, orexin) in two different brain regions resulted in significant suppression of protein expression without neurotoxicity. Expression was significantly suppressed at six to seven days post-administration, but returned to baseline levels within fourteen days. These results indicate that direct intracranial administration of vivo-morpholinos provides an effective means by which to suppress protein expression in the brain for one to two weeks.

Knocking down the transcript of protein kinase C-lambda modulates hypothalamic glutathione peroxidase, melanocortin receptor and neuropeptide Y gene expression in amphetamine-treated rats.

It has been reported that neuropeptide Y (NPY) contributes to the behavioral response of amphetamine (AMPH), a psychostimulant. The present study examined whether protein kinase C (PKC)-λ signaling was involved in this action. Moreover, possible roles of glutathione peroxidase (GP) and melanocortin receptor 4 (MC4R) were also examined. Rats were given AMPH daily for 4 days. Hypothalamic NPY, PKCλ, GP and MC4R were determined and compared. Pretreatment with α-methyl-para-tyrosine could block AMPH-induced anorexia, revealing that endogenous catecholamine was involved in regulating AMPH anorexia. PKCλ, GP and MC4R were increased with maximal response on Day 2 during AMPH treatment, which were concomitant with the decreases in NPY. cAMP response element binding protein (CREB) DNA binding activity was increased during AMPH treatment, revealing the involvement of CREB-dependent gene transcription. An interruption of cerebral PKCλ transcript could partly block AMPH-induced anorexia and partly reverse NPY, MC4R and GP mRNA levels to normal. These results suggest that PKCλ participates in regulating AMPH-induced anorexia via a modulation of hypothalamic NPY gene expression and that increases of GP and MC4R may contribute to this modulation. Our results provided molecular evidence for the regulation of AMPH-induced behavioral response.

Intra-amygdala injections of CREB antisense impair inhibitory avoidance memory: role of norepinephrine and acetylcholine.

Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed brain sites, through mechanisms that include the release of norepinephrine and acetylcholine within the amygdala. Thus, CREB antisense injections may affect memory by interfering with mechanisms of modulation, rather than storage, of memory. In the present experiment, rats received bilateral intra-amygdala infusions of CREB antisense (2 nmol/1 microL) 6 h prior to inhibitory avoidance training. In vivo microdialysis samples were collected from the right amygdala before, during, and following training. CREB antisense produced amnesia tested at 48 h after training. In addition, CREB antisense infusions dampened the training-related release of norepinephrine, and to a lesser extent of acetylcholine, in the amygdala. Furthermore, intra-amygdala infusions of the beta-adrenergic receptor agonist clenbuterol administered immediately after training attenuated memory impairments induced by intra-amygdala injections of CREB antisense. These findings suggest that intra-amygdala treatment with CREB antisense may affect processes involved in modulation of memory in part through interference with norepinephrine and acetylcholine neurotransmission in the amygdala.

Drug delivery systems: application of liposomal anti-tumor agents to neuroectodermal cancer treatment.

Disseminated neuroectoderma-derived tumors, mainly neuroblastoma in childhood and melanoma in the adulthood, are refractory to most current therapeutic regimens and hence the prognosis remains very poor. Preclinical research studies have indicated several agents that show promising therapeutic potential for these neoplasms. However, there appears to be a limitation to their in vivo applicability, mainly due to unfavorable pharmacokinetic properties that lead to insufficient drug delivery to the tumor or metastatic sites or to high systemic or organ-specific toxicity. In this scenario, the focus is on targeted cancer therapy. Encapsulating anticancer drugs in liposomes enables targeted drug delivery to tumor tissue and prevents damage to the normal surrounding tissue. Indeed, sterically stabilized liposomes have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. The identification of tumor-associated antigens and/or genes and the relative ease of manipulating the physicochemical features of liposome hold promise for the development of novel therapeutic strategies that selectively target tumor cells. Combined targeting is still investigated, especially the availability to simultaneously target and kill both the cancer cells and the tumor vasculature. Animal models make it possible to link molecular genetics and biochemistry information to the physiological basis of disease and are important predictive tools that offer a frontline testing system for studying the involvement of specific genes and the efficacy of novel therapeutics approaches. Relevant experimental models of human neuroblastoma and melanoma, which better reflect the tumor behavior in patients, are required to evaluate the effectiveness of the various targeted liposomal formulations and their possible systemic and organ-specific toxicity. The most multifunctional targeted liposomes are herein described, with primary attention on testing their efficacy in clinically relevant animal models for the treatment of neuroblastoma and melanoma.

Gene transfer of antisense B7.1 attenuates acute rejection against splenic allografts in rats.

Blockade of CD80-CD28 costimulatory pathway induces unresponsiveness of T cells to alloantigens and protects allografts against immune rejection in numerous animal models. The aim of this study was to investigate whether blocking expression of B7.1 (CD80) on donor splenocytes by an antisense technique protected splenic allografts against immune rejection. Splenic grafts from Wistar-Furth rats were intra-arterially transfused with an antisense B7.1 expression vector, before they were transplanted into Sprague-Dawley rats. The rats were sacrificed at scheduled times, and the splenic allografts histologically examined. Antisense gene transfer resulted in marked down-regulation of B7.1 in donor spleens, hyporesponsiveness of recipient T cells, and attenuated acute immune rejection against splenic allografts. No obvious damage to skin, liver, or gut due to graft-versus-host disease was detected in the recipients. In conclusion, blocking expression of B7.1 in donor spleens by antisense gene therapy represented a potential alloantigen-specific immunosuppressive strategy to inhibit acute rejection against splenic allografts.

Evaluation of basic amphipathic peptides for cellular delivery of antisense peptide nucleic acids.

Cellular permeation peptides have been used successfully for the delivery of a variety of cargoes across cellular membranes, including large hydrophilic biomolecules such as proteins, oligonucleotides, or plasmid DNA. For the present work, a series of short amphipathic peptides was designed to elucidate the structural requirements for efficient and nontoxic delivery of peptide nucleic acids (PNAs). On the basis of an idealized alpha-helical structure, the helical parameters were modulated systematically to yield peptides within a certain range of hydrophobicity and amphipathicity. The corresponding PNA conjugates were synthesized and characterized in terms of secondary structure, enzymatic stability, and antisense activity. The study revealed correlations between the physicochemical and biophysical properties of the conjugates and their biological activity and led to the development of potent peptide vectors for the cellular delivery of antisense PNAs. Two representative compounds were radiolabeled and evaluated for their biodistribution in healthy mice.

DNA-based therapeutics and DNA delivery systems: a comprehensive review.

The past several years have witnessed the evolution of gene medicine from an experimental technology into a viable strategy for developing therapeutics for a wide range of human disorders. Numerous prototype DNA-based biopharmaceuticals can now control disease progression by induction and/or inhibition of genes. These potent therapeutics include plasmids containing transgenes, oligonucleotides, aptamers, ribozymes, DNAzymes, and small interfering RNAs. Although only 2 DNA-based pharmaceuticals (an antisense oligonucleotide formulation, Vitravene, (USA, 1998), and an adenoviral gene therapy treatment, Gendicine (China, 2003), have received approval from regulatory agencies; numerous candidates are in advanced stages of human clinical trials. Selection of drugs on the basis of DNA sequence and structure has a reduced potential for toxicity, should result in fewer side effects, and therefore should eventually yield safer drugs than those currently available. These predictions are based on the high selectivity and specificity of such molecules for recognition of their molecular targets. However, poor cellular uptake and rapid in vivo degradation of DNA-based therapeutics necessitate the use of delivery systems to facilitate cellular internalization and preserve their activity. This review discusses the basis of structural design, mode of action, and applications of DNA-based therapeutics. The mechanisms of cellular uptake and intracellular trafficking of DNA-based therapeutics are examined, and the constraints these transport processes impose on the choice of delivery systems are summarized. Finally, the development of some of the most promising currently available DNA delivery platforms is discussed, and the merits and drawbacks of each approach are evaluated.

Peptide-mediated cell delivery: application in protein target validation.

Recent reports have suggested that conjugation of peptides, proteins and antisense to short highly basic peptides, such as TAT, antennapedia and transportan, results in their rapid translocation into cells. Importantly, these conjugates have been shown to exert actions in a number of animal models suggesting their general utility for the determination of protein function in vitro and in vivo.

Intraperitoneal injection of antisense peptide nucleic acids targeted to the mu receptor decreases response to morphine and receptor protein levels in rat brain.

To determine the effectiveness of peptide nucleic acids (PNAs) in vivo, we designed and synthesized PNAs antisense to the mu receptor, the molecular target of morphine for inducing antinociception. Responsiveness of rats to morphine and the levels of mu receptor expression after treatment was measured. We delivered intraperitoneal injections of antisense PNAs targeted to the mu receptor (AS-MOR), mismatch PNAs (AS-MOR MM), antisense PNAs targeted to the neurotensin receptor subtype 1 (AS-NTR1), or saline and then challenged the rats with 5 mg/kg morphine (intraperitonally) or neurotensin directly into the periaqueductal gray region of the brain. To avoid tolerance, separate groups of animals were tested at 24, 48, and 72 h post-PNA treatment. Only animals treated with the AS-MOR showed a reduction in their antinociceptive response to morphine. The lack of effect of morphine on the AS-MOR rats was profound at 24 and 48 h, but animals tested at 72 h were similar to control groups. At 24 h the AS-MOR rats had a significant 55% decrease in the levels of mu receptor in their periaqueductal gray region, while AS-MOR MM rats showed no significant change. Lastly, the AS-MOR rats continued to show a normal antinociceptive response to neurotensin. This study, therefore, provides additional support for the use of PNAs to target proteins within brain by systemically administered PNAs.

Decreased insulin-like growth factor-II/mannose 6-phosphate receptor expression enhances tumorigenicity in JEG-3 cells.

The insulin-like growth factor-II/mannose-6 phosphate receptor (IGF-II/M6PR) is believed to bind and degrade the potent mitogen IGF-II, a growth factor for many tumors. This receptor has been shown to be mutated and/or lost in a significant percentage of a variety of tumors, implying that it may act as a negative regulator of cell growth. In this study, we demonstrate that down-regulation of this receptor, mediated by antisense IGF-II/M6PR cDNA transfection into JEG-3 choriocarcinoma cells, results in increased growth rate in vitro and increased tumor growth rate in vivo. These findings demonstrate that a decrease in IGF-II/M6PR expression results in a growth advantage in JEG-3 cells and are consistent with the hypothesis that the IGF-II/M6PR is an inhibitor of tumor growth.

HMG-I(Y) recognizes base-unpairing regions of matrix attachment sequences and its increased expression is directly linked to metastatic breast cancer phenotype.

Base-unpairing regions (BURs) contain a specialized DNA context with an exceptionally high unwinding propensity, and are typically identified within various matrix attachment regions. A BUR affinity column was used to purify a doublet of Mr 20,000 proteins from human breast carcinoma cells. These proteins were identified as the high-mobility group (HMG) protein, HMG-I, and its splicing variant, HMG-Y. We show that HMG-I(Y) specifically binds BURs. Mutating BURs so as to abrogate their unwinding property greatly reduced their binding affinity to HMG-I(Y). Numerous studies have indicated that elevated HMG-I(Y) expression is correlated with more advanced cancers and with increased metastatic potential. We studied whether the expression of HMG-I(Y) responds to signaling through the heregulin (HRG)-erbB pathway and the extracellular matrix. HMG-I(Y) expression was increased in MCF-7 cells after stable transfection with an HRG expression construct that led cells to acquire estrogen independence and metastasizing ability. A high level of HMG-I(Y) expression was detected in metastatic MDA-MB-231 cells, but the expression was virtually diminished, and the metastasizing ability was lost after cells were stably transfected with an antisense HRG cDNA construct. HMG-I(Y) was also decreased in MDA-MB-231 cells when treated with a chemical inhibitor for matrix metalloproteinase-9 that led to a reduction of invasive capability in vitro. The level of HMG-I(Y) expression, therefore, is dynamically regulated in human breast cancer cells in response to varying types of signaling that affect metastatic ability, including the HRG-erbB pathway and those from the extracellular matrix.

Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood-brain barrier and specifically reduce gene expression.

Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood-brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i. p. delivery by crossing the normal blood-brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown in vivo to be a potentially effective therapeutic treatment.

Clinical aspects of intratumoral gene therapy.

One of the major obstacles to the development of gene therapy for cancer is our inability to deliver genes to all targets within the body. Thus, effective methodology does not exist to deliver a gene intravenously with the expectation that it will selectively localize within the target tumor, will not localize in other tissues and will be expressed efficiently. While one can take advantage of tissue-specific promoters to activate the gene only in a given target tissue, only a small fraction of the vector will be taken up in the target tissue and expressed. Consequently, since accessible local or regional tumor masses are a major problem in many cancers, there has been a strong emphasis on clinical trials in intratumoral and peritumoral gene delivery.

CNS drug design based on principles of blood-brain barrier transport.

Lipid-soluble small molecules with a molecular mass under a 400-600-Da threshold are transported readily through the blood-brain barrier in vivo owing to lipid-mediated transport. However, other small molecules lacking these particular molecular properties, antisense drugs, and peptide-based pharmaceuticals generally undergo negligible transport through the blood-brain barrier in pharmacologically significant amounts. Therefore, if present day CNS drug discovery programs are to avoid termination caused by negligible blood-brain barrier transport, it is important to merge CNS drug discovery and CNS drug delivery as early as possible in the overall CNS drug development process. Strategies for special formulation that enable drug transport through the blood-brain barrier arise from knowledge of the molecular and cellular biology of blood-brain barrier transport processes.

Antisense strategies and therapeutic applications.

The concepts underlying the antisense approach to disease therapy are discussed, and potential applications are examined. Antisense therapeutic agents bind to DNA or RNA sequences, blocking the synthesis of cellular proteins with unparalleled specificity. Transcription and translation are the two processes with which the agents interfere. There are three major classes of antisense agents: antisense sequences, commonly called antisense oligonucleotides; antigene sequences; and ribozymes. Antisense sequences are derivatives of nucleic acids that hybridize cytosolic messenger RNA (mRNA) sense strands through hydrogen bonding to complementary nucleic acid bases. Antigene sequences hybridize double-stranded DNA in the nucleus, forming triple helixes. Ribozymes, rather than inhibiting protein synthesis simply by binding to a single targeted mRNA, combine enzymatic processes with the specificity of antisense base pairing, creating a molecule that can incapacitate multiple targeted mRNAs. Antisense therapeutic agents are being investigated in vitro and in vivo for use in treating human immunodeficiency virus infection, hepatitis B virus infection, herpes simplex virus infection, papillomavirus infection, cancer, restenosis, rheumatoid arthritis, and allergic disorders. Although many results are preliminary, some are promising and have led to clinical trials. A major goal in developing methods of delivering antisense agents is to reduce their susceptibility to nucleases while retaining their ability to bind to targeted sites. Modification of the phosphodiester linkages in oligonucleotides can lend the sequences enzymatic stability without affecting their binding capacities. Carrier systems designed to protect the antisense structure and improve passage through the cell membrane include liposomes, water-soluble polymers, and nanoparticles. The pharmacokinetics of antisense agents are under investigation. Antisense therapeutic agents have the potential to become an integral part of medicinal regimens.