Cyclodextrin Prevents Abdominal Aortic Aneurysm via Activation of Vascular Smooth Muscle Cell Transcription Factor EB.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. METHODS: The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. RESULTS: We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-ß-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-ß-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. CONCLUSIONS: Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-ß-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model.

Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP-) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP-16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP-16 tumor revealed their proximity to known MLV common insertion site genes while maintaining the MLV IN TP- genotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TP- within chromatin profile states associated with weakly transcribed heterochromatin with fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TP- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein.

Development of Novel Adenoviral Vectors to Overcome Challenges Observed With HAdV-5-based Constructs.

Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5-mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes.

Reversibly cross-linked polyplexes enable cancer-targeted gene delivery via self-promoted DNA release and self-diminished toxicity.

Polycations often suffer from the irreconcilable inconsistency between transfection efficiency and toxicity. Polymers with high molecular weight (MW) and cationic charge feature potent gene delivery capabilities, while in the meantime suffer from strong chemotoxicity, restricted intracellular DNA release, and low stability in vivo. To address these critical challenges, we herein developed pH-responsive, reversibly cross-linked, polyetheleneimine (PEI)-based polyplexes coated with hyaluronic acid (HA) for the effective and targeted gene delivery to cancer cells. Low-MW PEI was cross-linked with the ketal-containing linker, and the obtained high-MW analogue afforded potent gene delivery capabilities during transfection, while rapidly degraded into low-MW segments upon acid treatment in the endosomes, which promoted intracellular DNA release and reduced material toxicity. HA coating of the polyplexes shielded the surface positive charges to enhance their stability under physiological condition and simultaneously reduced the toxicity. Additionally, HA coating allowed active targeting to cancer cells to potentiate the transfection efficiencies in cancer cells in vitro and in vivo. This study therefore provides an effective approach to overcome the efficiency-toxicity inconsistence of nonviral vectors, which contributes insights into the design strategy of effective and safe vectors for cancer gene therapy.

Galactosylated magnetic nanovectors for regulation of lipid metabolism based on biomarker-specific RNAi and MR imaging.

The specific delivery of ribonucleic acid (RNA) interfering molecules to disease-related cells is still a critical blockade for in vivo systemic treatment. Here, this study suggests a robust delivery carrier for targeted delivery of RNA-interfering molecules using galactosylated magnetic nanovectors (gMNVs). gMNVs are an organic-inorganic polymeric nanomaterial composed of polycationics and magnetic nanocrystal for delivery of RNA-interfering molecules and tracking via magnetic resonance (MR) imaging. In particular, the surface of gMNVs was modified by galactosylgluconic groups for targeted delivering to asialoglycoprotein receptor (ASGPR) of hepatocytes. Moreover, the small interfering RNAs were used to regulate target proteins related with low-density lipoprotein level and in vivo MR imaging was conducted for tracking of nanovectors. The obtained results show that the prepared gMNVs demonstrate potential as a systemic theragnostic nanoplatform for RNA interference and MR imaging.

Long-term toxicity study of rAd5-hTERTC27 in SD rats and Cynomolgus monkeys by intravenous injection.

rAd5-hTERTC27, a replication-defective adenovirus vector carrying hTERTC27, has been proposed for possible use against hepatocellular carcinoma (HCC). In this study, we investigated the long-term toxicity of rAd5-hTERTC27 in SD rats and Cynomolgus monkeys. rAd5-hTERTC27 was administered intravenously once a week for 13 weeks followed by a one-month recovery period. As of 4 months, all animals displayed overall good health. Anti-adenoviral antibodies emerged in a dose-independent manner. The levels of complement components, C3 and C4, in the rAd5-hTERTC27 middle-dose and high-dose groups and C4 in the rAd5-EGFP group increased significantly after the 2nd treatment in monkeys. Slight-mild pathological changes of the liver occurred only in the rAd5-hTERTC27 high-dose group (2/16) in rats and not in any other group in either rats or monkeys. With the increase of the dose, the incidence of lymphocyte depletion in the spleen of rats and reactive hyperplasia of the splenic corpuscle in monkeys increased. However, the changes in the liver and spleen were reversible. Given the above data, intravenous administration of rAd5-hTERTC27 (up to 4×10(10)VP/kg in rats and 0.9×10(10)VP/kg in monkeys) appears to be well-tolerated, providing support for its potentially safe use in clinical trials for the treatment of HCC.

Impaired neurotransmission caused by overexpression of α-synuclein in nigral dopamine neurons.

We used in vivo amperometry to monitor changes in synaptic dopamine (DA) release in the striatum induced by overexpression of human wild-type α-synuclein in nigral DA neurons, induced by injection of an adeno-associated virus type 6 (AAV6)-α-synuclein vector unilaterally into the substantia nigra in adult rats. Impairments in DA release evolved in parallel with the development of degenerative changes in the nigrostriatal axons and terminals. The earliest change, seen 10 d after vector injection, was a marked, ≈50%, reduction in DA reuptake, consistent with an early dysfunction of the DA transporter that developed before any overt signs of axonal damage. At 3 wk, when the first signs of axonal damage were observed, the amount of DA released after a KCl pulse was reduced by 70-80%, and peak DA concentration was delayed, indicating an impaired release mechanism. At later time points, 8-16 wk, overall striatal innervation density was reduced by 60-80% and accompanied by abundant signs of axonal damage in the form of α-synuclein aggregates, axonal swellings, and dystrophic axonal profiles. At this stage DA release and reuptake were profoundly reduced, by 80-90%. The early changes in synaptic DA release induced by overexpression of human α-synuclein support the idea that early predegenerative changes in the handling of DA may initiate, and drive, a progressive degenerative process that hits the axons and terminals first. Synaptic dysfunction and axonopathy would thus be the hallmark of presymptomatic and early-stage Parkinson disease, followed by neuronal degeneration and cell loss, characteristic of more advanced stages of the disease.

Influence of oligospermines architecture on their suitability for siRNA delivery.

Spermines are naturally abundant polyamines that partially condense nucleic acids and exhibit the proton-sponge effect in an acidic environment. However, spermines show a limited efficiency for transfecting nucleic acids because of their low molecular weight. Therefore, spermines need to be modified to be used as nonviral vectors for nucleic acids. Here, we synthesized linear bisspermine as well as a linear and dendritic tetraspermine with different molecular architectures. These oligospermines were self-assembled into polyplexes with siRNA. The structure-activity relationship of the oligospermines was evaluated in terms of their efficiency for delivering siRNA into a nonsmall cell lung carcinoma cell line. Oligospermines displayed minimal cytotoxicity but efficient siRNA condensation and showed better stability against polyanions than polyethylenimine. The morphology of the polyplexes was strongly affected by the oligospermine architecture. Linear tetraspermine/siRNA polyplexes showed the best gene-silencing efficiency among the oligospermines tested at both the mRNA and protein expression levels, indicating the most favorable structure for siRNA delivery.

Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice.

Neuropeptide Y (NPY) exerts anxiolytic- and antidepressant-like effects in rodents that appear to be mediated via Y1 receptors. Gene therapy using recombinant viral vectors to induce overexpression of NPY in the hippocampus or amygdala has previously been shown to confer anxiolytic-like effect in rodents. The present study explored an alternative and more specific approach: overexpression of Y1 receptors. Using a recombinant adeno-associated viral vector (rAAV) encoding the Y1 gene (rAAV-Y1), we, for the first time, induced overexpression of functional transgene Y1 receptors in the hippocampus of adult mice and tested the animals in anxiety- and depression-like behavior. Hippocampal Y1 receptors have been suggested to mediate seizure-promoting effect, so the effects of rAAV-induced Y1 receptor overexpression were also tested in kainate-induced seizures. Y1 receptor transgene overexpression was found to be associated with modest anxiolytic-like effect in the open field and elevated plus maze tests, but no effect was seen on depression-like behavior using the tail suspension and forced swim tests. However, the rAAV-Y1 vector modestly aggravated kainate-induced seizures. These data indicate that rAAV-induced overexpression of Y1 receptors in the hippocampus could confer anxiolytic-like effect accompanied by a moderate proconvulsant adverse effect. Further studies are clearly needed to determine whether Y1 gene therapy might have a future role in the treatment of anxiety disorders.

Intracardiac injection of a capsid-modified Ad5/35 results in decreased heart toxicity when compared to standard Ad5.

BACKGROUND: Clinical gene therapy trials for cardiovascular diseases have demonstrated the crucial role of efficient gene delivery and transfection technologies in achieving clinically relevant results. We hypothesized that the use of tropism-modified adenoviruses would improve transduction efficacy and to this end we analyzed the transduction efficiency and toxicity of standard Ad5 and tropism-modified Ad5/35 in combination with ultrasound-guided intramyocardial gene delivery. METHODS: Ultrasound-guided intracardiac injections were used to deliver 1 × 10(10) pfu/ml Ad5-lacZ and Ad5/35-lacZ vectors into mouse left ventricle wall. Since Ad5/35 uses human CD46 as its primary receptor, we used transgenic hCD46Ge mice expressing human CD46 at levels comparable to man. Mice were sacrificed 6 or 14 days post-injection and immunohistochemistry and X-gal staining were used to detect transgene and viral receptor expression. Virus-induced cardiac toxicity was evaluated by a pathologist. RESULTS: The intramyocardial injection was well tolerated and both Ad5-lacZ and Ad5/35-lacZ were able to give robust transgene expression after a single injection. Interestingly, while Ad5-lacZ was able to generate greater transgene expression than Ad5/35-lacZ, it also evoked more severe tissue damage with large areas of interstitial inflammatory cell infiltration and myocyte necrosis. CONCLUSIONS: Ultrasound-guided intramyocardial injection is an effective and safe way to deliver vectors to the heart. The observed severe tissue damage of Ad5-lacZ greatly undermines the efficient transgene expression and suggests that Ad5/35 capsid modification can result in safer adenoviral vectors for cardiovascular gene therapy, although at the cost of some vector transduction efficacy.

Cationic carriers of genetic material and cell death: a mitochondrial tale.

Central to gene therapy technology has been the use of cationic polymers as vectors for DNA and RNA (polyfectins). These have been presumed to be safer than viral systems which, for example, have been found to switch on oncogenes. Two key polycations that have been intensively researched for use as synthetic vectors are poly(ethylenimine) and poly(L-lysine). A frequent stumbling block with these polyfectins is that long-term gene expression in cell lines has not been achieved. Recently it has transpired that both of these polycations can induce mitochondrially mediated apoptosis. It is the aim of this review to discuss the mechanisms behind the observed polycation toxicity including roles for little studied cellular organelles in the process such as the lysosome and endoplasmic reticulum.

Development challenges associated with rAAV-based gene therapies.

The number of gene therapies in development continues to increase, as they represent a novel method to treat, and potentially cure, many diseases. Gene therapies can be conducted with an in vivo or ex vivo approach, to cause gene augmentation, gene suppression, or genomic editing. Adeno-associated viruses are commonly used to deliver gene therapies, but their use is associated with several manufacturing, nonclinical and clinical challenges. As these challenges emerge, regulatory agency expectations continue to evolve. Following administration of rAAV-based gene therapies, nonclinical toxicities may occur, which includes immunogenicity, hepatotoxicity, neurotoxicity, and the potential risks for insertional mutagenesis and subsequent tumorgenicity. The mechanism for these findings and translation into the clinical setting are unclear at this time but have influenced the nonclinical studies that regulatory agencies are increasingly requesting to support clinical trials and marketing authorizations. These evolving regulatory expectations and toxicities, as well as future nonclinical considerations, are discussed herein.

Efficient, long-term hepatic gene transfer using clinically relevant HDAd doses by balloon occlusion catheter delivery in nonhuman primates.

Helper-dependent adenoviral vectors (HDAd) are devoid of all viral coding sequences and are thus an improvement over early generation Ad because they can provide long-term transgene expression in vivo without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic intravenous injection, and this unfortunately results in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver HDAd into the liver of nonhuman primates. Briefly, a balloon occlusion catheter was percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow. HDAd was injected directly into the occluded liver via a percutaneously placed hepatic artery catheter. Compared to systemic vector injection, this approach resulted in substantially higher hepatic transduction efficiency using clinically relevant low vector doses and was accompanied by mild-to-moderate acute but transient toxicities. Transgene expression was sustained for up to 964 days. These results suggest that our minimally invasive method of delivery can significantly improve the vector's therapeutic index and may be a first step toward clinical application of HDAd for liver-directed gene therapy.

Pre-existing immunity and passive immunity to adenovirus 5 prevents toxicity caused by an oncolytic adenovirus vector in the Syrian hamster model.

We have used Syrian hamsters to examine the role of pre-existing immunity to adenovirus (Ad) 5 in the toxicity of the oncolytic Ad vector INGN 007. Groups of hamsters were or were not immunized with Ad5. Half the hamsters were immunosuppressed using cyclophosphamide (CP), then injected intravenously (i.v.) with 3x the maximum tolerated dose (MTD) of INGN 007 (in immunocompetent hamsters), and toxicity and vector replication in the liver were quantitated. In nonimmunized immunocompetent hamsters, toxicity was observed early but the hamsters recovered by day 6 after vector injection. In nonimmunized immunosuppressed hamsters, the vector was lethal by 3 days. Pre-existing neutralizing antibody (NAb) prevented liver infection and hepatotoxicity in both immunocompetent and immunosuppressed hamsters. In another study, passive immunization of immunosuppressed hamsters 1 day before a lethal dose (1x MTD) of INGN 007 prevented liver infection and replication, but immunization 1 day after vector administration was barely effective. When immunosuppressed hamsters were passively immunized 1 day after injection of 1/3rd the MTD of INGN 007, then significant protection was observed against liver infection and toxicity. Therefore, serum NAb are sufficient to prevent oncolytic Ad vector liver infection and toxicity. We saw no evidence that pre-existing immunity was associated with increased vector toxicity.

Polymeric micelles comprising stearic acid-grafted polyethyleneimine as nonviral gene carriers.

Non-viral vectors composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, or improve the insufficient transfection efficiency of lower molecular weight PEI. In this study, 10 k PEI was modified by grafting stearic acid (SA) and formulated to polymer micelles with positive surface charge and evaluated for pDNA delivery. The amine group on PEI was crosslinked with the carboxylic group of stearic acid by 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as linker. PEI-SA micelles were then prepared using oil in water (o/w) solvent evaporation method. The success of PEI-SA conjugation structure was confirmed with 1H NMR. The average diameter and zeta potential determined by photon correlation spectroscopy was 149.6 +/- 1.2 nm and 64.1 +/- 1.5 mV, respectively. These self-assemble positive charge micelles showed effective binding to pDNA for transfection. PEI-SA micelles exhibited lower cytotoxicity compared to that of PEI only, while flow cytometry analysis revealed PEI-SA/pEGFP complex provided 62% high EGFP expression. Luciferase activity also showed high transfection efficiency of PEI-SA micelles for weight ratio above 4.5 that was comparable to PEI only. These results demonstrated that stearic acid grafted PEI micelles can provide high transfection efficiency comparable to unmodified PEI, and exhibit low cytotoxicity. Stearic acid grafted PEI micelles can be promising polymer carriers in genetic therapy.

[Impact of polyamidoamine dendrimer liposome on the cellular uptake and cytotoxicity of colonic cancer cells].

OBJECTIVE: To evaluate the effects of polyamidoamine dendrimer (PAMAM) liposome as gene carriers on the cellular uptake and its cytotoxicity in colonic cancer cell. METHODS: The liposome modified PAMAM was synthesized with liposome and polyamidoamine dendrimer. Plasmid PEGFP-N1 was mixed with the liposome-modified PAMAM or unmodified PAMAM to form nanoparticle complexes. The shape and size of the nanoparticle complexes were observed by transmission electron microscope and the zeta potential was measured by analytical tool. The encapsulating efficiency was determined by ultraviolet spectrophotometer in centrifuging method. After the cell lines SW620 (colonic cancer cell), MCF-7 (breast cancer cell), ECV304 (vascular endothelial cell) were transfected by the two kinds of PAMAM nanoparticle complexes, the flow cytometry was used to determine the uptake of enhanced green fluorescent protein (EGFP) gene. The cytotoxicity of PAMAM liposome nanoparticles and PAMAM nanoparticles was evaluated by MTT assay. RESULTS: The diameter of liposome modified PAMAM complex was (192 ± 16) nm, and that of PAMAM complex was (189 ± 19) nm (P > 0.05); and the zeta potential of liposome modified PAMAM complex was higher than that of PAMAM complex [(42 ± 7) mV vs. (32 ± 7) mV, P < 0.05]. There was no significant difference in envelopment rate between the two groups [(82 ± 7)% vs. (84 ± 6)%, P > 0.05]. After the colonic cancer cell line SW620 was transfected with the two kinds of PAMAM nanoparticle complexes, the cellular uptake of the cells with the liposome-modified PAMAM complex was significantly higher than that of the cell with PAMAM complex (P < 0.05). The cellular survival rate of the cell lines with liposome-modified PAMAM complex was significantly higher than that of cell lines with PAMAM complex (P < 0.05). CONCLUSION: The liposome modified PAMAM can improve gene transfection efficiency and suppress its cytotoxicity.

Fatty acid-spermine conjugates as DNA carriers for nonviral in vivo gene delivery.

The lack of efficient in vivo gene delivery is a well-known shortcoming of nonviral delivery vectors, in particular of chemical vectors. We developed a series of novel nonviral carriers for plasmid-based in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with synthetic derivatives of naturally occurring molecules-fatty acid-spermine conjugates (or lipospermines). We tested the ability of these fatty acid conjugates to interact with plasmid DNA (pDNA) and found that they formed DNA nanocomplexes, which are protected from DNase I degradation. This protection was shown to directly correlate with the length of the aliphatic component. However, this increase in the length of the hydrocarbon chain resulted in increased toxicity. The cationic lipids used for transfection typically have a C(16) and C(18) hydrocarbon chain. Interestingly, toxicity studies, together with further characterization studies, suggested that the two most suitable candidates for in vivo delivery are those with the shortest hydrocarbon chain, butanoyl- and decanoylspermine. Morphological characterization of DNA nanocomplexes resulting from these lipospermines showed the formation of a homogenous population, with the diameter ranging approximately from 40 to 200 nm. Butanoylspermine was found to be the most promising carrier from this series, resulting in a significantly increased gene expression, in relation to naked plasmid, in both tissues herein targeted (dermis and M. tibialis anterior). Thus, we established a correlation between the in vitro properties of the ensuing DNA nanocarriers and their efficient in vivo gene expression.

A preclinical assessment of the safety and biodistribution of an adenoviral vector containing the herpes simplex virus thymidine kinase gene (Cerepro) after intracerebral administration.

BACKGROUND: Cerepro (sitimagene ceradenovec) is an adenoviral vector containing herpes simplex virus thymidine kinase gene (HSV-tk), which is being developed for the treatment of high-grade glioma with oral ganciclovir (GCV). The nonclinical safety and biodistribution of Cerepro were assessed following intravenous (i.v.) or intracerebral (i.c.) injection. METHODS: Crl : WI(GLX/BRL/Han) rats (n = 198) were injected i.c. or i.v. with Cerepro or vehicle control, with GCV by intraperitoneal (i.p.) injection to selected groups. Safety was assessed by observation of animal behaviour and post mortem histology. Antibody response was assessed, and biodistribution measured using the quantitative polymerase chain reaction (PCR) and reverse transcriptase-PCR in blood and tissues. RESULTS: Following i.v. or i.c. injection, there was no antibody response and no effect on behaviour, body weight, food consumption or haematological and clinical chemistry parameters. Minor needle track changes were observed in control and Cerepro-i.c. injection groups. Transient myeloid hyperplasia was observed in five of the 24 animals in the i.v. injection group and spleen weight increased in both the i.c. and i.v. groups. Cerepro was detected in the brain and at low levels in blood and spleen following i.c. injection, decreasing with time. Following i.v. injection, Cerepro was detected in viscera and blood, decreasing with time. Transcription of Cerepro was detected in the brain following i.c. injection, with lower levels in spleen; following i.v. injection, transcription was seen in viscera. Germline integration was not seen. CONCLUSIONS: Intracerebral injection of Cerepro is safe and produces a high level of transgene expression in the brain, with limited biodistribution.

Safe and sustained overexpression of functional apolipoprotein A-I/high-density lipoprotein in apolipoprotein A-I-null mice by muscular adeno-associated viral serotype 8 vector gene transfer.

High levels of high-density lipoprotein (HDL) have protective effects against atherosclerosis and cardiovascular diseases. The postulated mechanism of action for these benefits is an enhanced reverse cholesterol transport. Apolipoprotein A-I (ApoA-I) is the major protein of HDL. The clinical benefits of raising ApoA-I/HDL have been clearly established by clinical and epidemiological studies. Despite these observations, there are not very effective pharmacological means for raising HDL. ApoA-I gene delivery by viral vectors seems a promising strategy to raise ApoA-I/HDL levels. Sustained gene expression in animals and humans has been attained using adeno-associated viral (AAV) vectors. The aim of the present study was to determine the efficiency, safety, and biological activity of human ApoA-I intramuscularly delivered using an AAV vector in mice. AAV serotype 8 vectors encoding for human ApoA-I transgene were administered intraportally and intramuscularly in ApoA-I- deficient animals. ApoA-I levels were measured every 2 weeks post administration. The effectiveness of the generated HDL was tested in vitro in cholesterol-loaded macrophages. The administration of the vectors resulted in a significant and sustained increase in ApoA-I and HDL plasma levels for up to 16 weeks at similar extent by both routes of administration. Activity of the generated HDL in removal of cholesterol from cholesterol-loaded macrophages was similar in both groups. Our data suggest that intramuscular AAV8-mediated gene transfer of human ApoA-I results in a significant and maintained increase in ApoA-I and functional HDL.

Physiological promoters reduce the genotoxic risk of integrating gene vectors.

The possible activation of cellular proto-oncogenes as a result of clonal transformation is a potential limitation in a therapeutic approach involving random integration of gene vectors. Given that enhancer promiscuity represents an important mechanism of insertional transformation, we assessed the enhancer activities of various cellular and retroviral promoters in transient transfection assays, and also in a novel experimental system designed to measure the activation of a minigene cassette contained in stably integrating retroviral vectors. Retroviral enhancer-promoters showed a significantly greater potential to activate neighboring promoters than did cellular promoters derived from human genes, elongation factor-1alpha (EF1alpha) and phosphoglycerate kinase (PGK). Self-inactivating (SIN) vector design reduced but did not abolish enhancer interactions. Using a recently established cell culture assay that detects insertional transformation by serial replating of primary hematopoietic cells, we found that SIN vectors containing the EF1alpha promoter greatly decrease the risk of insertional transformation. Despite integration of multiple copies per cell, activation of the crucial proto-oncogene Evi1 was not detectable when using SIN-EF1alpha vectors. On the basis of several quantitative indicators, the decrease in transforming activity was highly significant (more than tenfold, P < 0.01) when compared with similarly designed vectors containing a retroviral enhancer-promoter with or without a well-characterized genetic insulator core element. In this manner, the insertional biosafety of therapeutic gene vectors can be greatly enhanced and proactively evaluated in sensitive cell-based assays.