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Assessment of anti-adeno-associated virus (AAV) antibodies in patients prior to systemic gene therapy administration is an important consideration regarding efficacy and safety of the therapy. Approximately 30%–60% of individuals have pre-existing anti-AAV antibodies. Seroprevalence is impacted by multiple factors, including geography, age, capsid serotype, and assay type. Anti-AAV antibody assays typically measure (1) transduction inhibition by detecting the neutralizing capacity of antibodies and non-antibody neutralizing factors, or (2) total anti-capsid binding antibodies, regardless of neutralizing activity. Presently, there is a paucity of head-to-head data and standardized approaches associating assay results with clinical outcomes. In addition, establishing clinically relevant screening titer cutoffs is complex. Thus, meaningful comparisons across assays are nearly impossible. Although complex, establishing screening assays in routine clinical practice to identify patients with antibody levels that may impact favorable treatment outcomes is achievable for both transduction inhibition and total antibody assays. Formal regulatory approval of such assays as companion diagnostic tests will confirm their suitability for specific recombinant AAV gene therapies. This review covers current approaches to measure anti-AAV antibodies in patient plasma or serum, their potential impact on therapeutic safety and efficacy, and investigative strategies to mitigate the effects of pre-existing anti-AAV antibodies in patients.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3Jmice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

Neuronopathic glycosphingolipidoses are a sub-group of lysosomal storage disorders for which there are presently no effective therapies. Here, we evaluated the potential of substrate reduction therapy (SRT) using an inhibitor of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide (GL1) and related glycosphingolipids. The substrates that accumulate in Sandhoff disease (e.g., ganglioside GM2 and its nonacylated derivative, lyso-GM2) are distal to the drug target, GCS. Treatment of Sandhoff mice with a GCS inhibitor that has demonstrated CNS access (Genz-682452) reduced the accumulation of GL1 and GM2, as well as a variety of disease-associated substrates in the liver and brain. Concomitant with these effects was a significant decrease in the expression of CD68 and glycoprotein non-metastatic melanoma B protein (Gpnmb) in the brain, indicating a reduction in microgliosis in the treated mice. Moreover, using in vivoimaging, we showed that the monocytic biomarker translocator protein (TSPO), which was elevated in Sandhoff mice, was normalized following Genz-682452 treatment. These positive effects translated in turn into a delay (∼28 days) in loss of motor function and coordination, as measured by rotarod latency, and a significant increase in longevity (∼17.5%). Together, these results support the development of SRT for the treatment of gangliosidoses, particularly in patients with residual enzyme activity.

For inherited genetic diseases, fetal gene therapy offers the potential of prophylaxis against early, irreversible and lethal pathological change. To explore this, we studied neuronopathic Gaucher disease (nGD), caused by mutations in GBA. In adult patients, the milder form presents with hepatomegaly, splenomegaly and occasional lung and bone disease; this is managed, symptomatically, by enzyme replacement therapy. The acute childhood lethal form of nGD is untreatable since enzyme cannot cross the blood–brain barrier. Patients with nGD exhibit signs consistent with hindbrain neurodegeneration, including neck hyperextension, strabismus and, often, fatal apnea1. We selected a mouse model of nGD carrying a loxP-flanked neomycin disruption of Gbaplus Cre recombinase regulated by the keratinocyte-specific K14 promoter. Exclusive skin expression of Gbaprevents fatal neonatal dehydration. Instead, mice develop fatal neurodegeneration within 15 days2. Using this model, fetal intracranial injection of adeno-associated virus (AAV) vector reconstituted neuronal glucocerebrosidase expression. Mice lived for up to at least 18 weeks, were fertile and fully mobile. Neurodegeneration was abolished and neuroinflammation ameliorated. Neonatal intervention also rescued mice but less effectively. As the next step to clinical translation, we also demonstrated the feasibility of ultrasound-guided global AAV gene transfer to fetal macaque brains.

The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.

Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration.

As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290that generates a cryptic splice donor site. The large size of the CEP290gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290splice mutation. We generated a cellular model of LCA10 by introducing the CEP290splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10.

What is this article about?This is a summary of a review article about gene therapy. Review articles summarize many previously published scientific articles. The review was about how a common viruscould change how one type of gene therapy works. It also discussed the importance of antibody testing for people who may receive gene therapy. The original review was published in Molecular Therapyin 2023.Gene therapy is a type of medical treatment. The goal of a specific gene therapy is to treat a specific genetic condition. A genetic condition is a health problem caused by a variation (change or mutation) in a geneor genes. Genes are instructions that tell the body how to grow and work.Some genetic conditions can cause rare diseases. A rare disease affects only a very small percentage of people. Gene therapy may help people with some rare diseases because it aims to correct the root cause of the condition instead of just treating symptoms.Gene therapies work by getting into the cellsin a person's body. Many gene therapies are modeled after viruses because viruses are good at getting into the body.One of the viruses used in gene therapy is called adeno-associated virus(AAVfor short). This naturally occurring virus infects many people. People can get infected without realizing it because AAV doesn't typically make a person sick.After being infected with a virus, people make antibodiesto fight off that virus. These antibodies can prevent another infection. Antibodies that fight off natural AAV may prevent gene therapy based on AAV from working as it should.A common reason why people may not be able to receive gene therapy is because they have antibodies against AAV. This means that some people with some genetic conditions or rare diseases may not be able to benefit from gene therapy. Researchers are trying to understand how antibodies against AAV can prevent gene therapy from working. They are also studying what side effectsthey may cause. This research is important for people with a genetic condition or rare disease that may be treated with gene therapy.What conclusions did we draw?People who have a certain level of antibodies against AAV might not be able to take part in gene therapy clinical trials. They might not be able to receive AAV-based gene therapies that have been approved for commercial use by a regulatory agency such as the FDA (Food and Drug Administration) or EMA (European Medicines Agency).For most gene therapies, people need to be tested for antibodies against AAV.There are two different types of tests that measure if someone has antibodies against AAV.Researchers are looking at ways to lower the effects of antibodies against AAV. This research could allow more people to have gene therapy in the future.

Gaucher disease (GD) is caused by a deficiency of glucocerebrosidase and the consequent lysosomal accumulation of unmetabolized glycolipid substrates. Enzyme-replacement therapy adequately manages the visceral manifestations of nonneuronopathic type-1 Gaucher patients, but not the brain disease in neuronopathic types 2 and 3 GD. Substrate reduction therapy through inhibition of glucosylceramide synthase (GCS) has also been shown to effectively treat the visceral disease. Here, we evaluated the efficacy of a novel small molecule inhibitor of GCS with central nervous system (CNS) access (Genz-682452) to treat the brain disease. Treatment of the conduritol β epoxide-induced mouse model of neuronopathic GD with Genz-682452 reduced the accumulation of liver and brain glycolipids (>70% and >20% respectively), extent of gliosis, and severity of ataxia. In the genetic 4L;C* mouse model, Genz-682452 reduced the levels of substrate in the brain by >40%, the extent of gliosis, and paresis. Importantly, Genz-682452-treated 4L;C* mice also exhibited an ~30% increase in lifespan. Together, these data indicate that an orally available antagonist of GCS that has CNS access is effective at attenuating several of the neuropathologic and behavioral manifestations associated with mouse models of neuronopathic GD. Therefore, Genz-682452 holds promise as a potential therapeutic approach for patients with type-3 GD.

•The objective of this research was to establish a method for the validation of commercially F1 hybrid groupers developed for commercial aquaculture.•The complete mitochondrial genome (mtDNA) of the E. lanceolatuswas sequenced.•The sequenced genomic fragments were assembled, annotated and deposited at the NCBI GenBank.•Locus specific primers were developed and applied to test the F1 hybrids and related species of groupers in order to test for cross-amplification patterns.•The amplification profiles confirmed the maternal inheritance of mtDNA in the grouper F1 hybrid.•This method can be applied to validate hybridization events in the case of inter-specific grouper hybrids.•Analysis of the mtDNA sequence revealed a distinct variation in the D-loop (control) region.

Lung delivery of plasmid DNA encoding the CFTRgene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTRgene therapy in patients with cystic fibrosis.

Supplemental Digital Content is available in the text.

Fabry disease is an X-linked lysosomal storage disease caused by deficient activity of α-galactosidase A and the resultant systemic accumulation of globotrioasylceramide (GL-3) and related glycolipids. α-Galactosidase A gene knockout (GlaKO) mice have no α-galactosidase A activity and progressively accumulate GL-3 in tissues and fluids, similarly to FD patients. The nature and temporal effects of the progressive substrate accumulation on tissue histology in these mice have not previously been characterized. Here, we report the pathology of young to old (3 to 17 months old) GlaKO mice and compare these changes with those in strain-matched control animals. GlaKO mice accumulated GL-3 in various tissues and fluids with age. Lysosomal GL-3 inclusions increased with age in multiple cell types, including renal epithelial, intestinal, and vascular smooth muscle cells, and neurons in trigeminal and dorsal root ganglia, as detected by light and electron microscopy. However, unlike the case for male FD patients with the type 1 classic phenotype, GL-3 inclusions were not detected in vascular endothelial cells or cardiomyocytes. The histological changes in GlaKO mice better resemble the type 2 later-onset phenotype observed in patients with residual α-galactosidase A activity. GL-3 accumulation in the small intestine and sensory ganglia of GlaKO mice provides a model for study of enteropathy and neuropathy in Fabry disease.

Over the past several years, considerable progress has been made in the development of gene therapy as a therapeutic strategy for a variety of inherited metabolic diseases, including neuropathic lysosomal storage disorders (LSDs). The premise of gene therapy for this group of diseases is borne of findings that genetic modification of a subset of cells can provide a more global benefit by virtue of the ability of the secreted lysosomal enzymes to effect cross-correction of adjacent and distal cells. Preclinical studies in small and large animal models of these disorders support the application of either a direct in vivo approach using recombinant adeno-associated viral vectors or an ex vivo strategy using lentiviral vector-modified hematopoietic stem cells to correct the neurological component of these diseases. Early clinical studies utilizing both approaches have begun or are in late-stage planning for a small number of neuropathic LSDs. Although initial indications from these studies are encouraging, it is evident that second-generation vectors that exhibit a greater safety profile and transduction activity may be required before this optimism can be fully realized. Here, I review recent progress and the remaining challenges to treat the neurological aspects of various LSDs using this therapeutic paradigm.

Background: Huntington's disease (HD) is a neurological disorder caused by mutations in the huntingtin (HTT) gene, the product of which leads to selective and progressive neuronal cell death in the striatum and cortex. Transcriptional dysregulation has emerged as a core pathologic feature in the CNS of human and animal models of HD. It is still unclear whether perturbations in gene expression are a consequence of the disease or importantly, contribute to the pathogenesis of HD. Objective: To examine if transcriptional dysregulation can be ameliorated with antisense oligonucleotides that reduce levels of mutant Htt and provide therapeutic benefit in the YAC128 mouse model of HD. Methods: Quantitative real-time PCR analysis was used to evaluate dysregulation of a subset of striatal genes in the YAC128 mouse model. Transcripts were then evaluated following ICV delivery of antisense oligonucleotides (ASO). Rota rod and Porsolt swim tests were used to evaluate phenotypic deficits in these mice following ASO treatment. Results: Transcriptional dysregulation was detected in the YAC128 mouse model and appears to progress with age. ICV delivery of ASOs directed against mutant Htt resulted in reduction in mutant Htt levels and amelioration in behavioral deficits in the YAC128 mouse model. These improvements were correlated with improvements in the levels of several dysregulated striatal transcripts. Conclusions: The role of transcriptional dysregulation in the pathogenesis of Huntington's disease is not well understood, however, a wealth of evidence now strongly suggests that changes in transcriptional signatures are a prominent feature in the brains of both HD patients and animal models of the disease. Our study is the first to show that a therapeutic agent capable of improving an HD disease phenotype is concomitantly correlated with normalization of a subset of dysregulated striatal transcripts. Our data suggests that correction of these disease-altered transcripts may underlie, at least in part, the therapeutic efficacy shown associated with ASO-mediated correction of HD phenotypes and may provide a novel set of early biomarkers for evaluating future therapeutic concepts for HD.

Respiratory function is the main cause of mortality in patients with Duchenne muscular dystrophy (DMD). Elevated levels of TGF-β play a key role in the pathophysiology of DMD. To determine whether therapeutic attenuation of TGF-β signaling improves respiratory function, mdxmice were treated from 2 weeks of age to 2 months or 9 months of age with either 1D11 (a neutralizing antibody to all three isoforms of TGF-β), losartan (an angiotensin receptor antagonist), or a combination of the two agents. Respiratory function was measured in nonanesthetized mice by plethysmography. The 9-month-old mdxmice had elevated Penh values and decreased breathing frequency, due primarily to decreased inspiratory flow rate. All treatments normalized Penh values and increased peak inspiratory flow, leading to decreased inspiration times and breathing frequency. Additionally, forelimb grip strength was improved after 1D11 treatment at both 2 and 9 months of age, whereas, losartan improved grip strength only at 2 months. Decreased serum creatine kinase levels (significant improvement for all groups), increased diaphragm muscle fiber density, and decreased hydroxyproline levels (significant improvement for 1D11 only) also suggested improved muscle function after treatment. For all endpoints, 1D11 was equivalent or superior to losartan; coadministration of the two agents was not superior to 1D11 alone. In conclusion, TGF-β antagonism may be a useful therapeutic approach for treating DMD patients.

Lysosomal storage disorders (LSD) are a group of approximately 40 genetic diseases that are caused by the deficiency of one or more lysosomal enzymes. The incidence of LSD is estimated to be approximately 1 in 7500 live births, which makes this one of the more prevalent groups of genetic diseases in humans. The loss in enzymatic activity leads to the accumulation of undegraded substrates within lysosomes, resulting in distension of the organelle and subsequent cellular malfunction. Although palliative treatments such as enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) have been shown to be effective for some of the LSD such as Gaucher, Fabry and MPS I, they are not available as yet, or ineffective, for a large number of other LSD patients. To fulfill this unmet medical need, gene therapy is being considered as an alternate or adjunctive therapy for this group of disorders. A goal of gene therapy for LSD is to introduce a normal copy of the DNA for the lysosomal enzyme into a depot organ such as the liver or muscle with the intent that this will lead to the sustained production and reconstituion of therapeutic levels of the enzyme in the affected tissues. Here, we review the utility of various gene therapy strategies under consideration for the treatment of the LSD, including viral and non-viral gene transfer approaches, as well as stem cell transplantation.

To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid α-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.

The mammalian innate immune system has the ability to recognise and direct a response against incoming foreign DNA. The primary signal that triggers this response is unmethylated CpG motifs present in the DNA sequence of various disease-causing pathogens. These motifs are rare in vertebrate DNA, but abundant in bacterial and some viral DNAs. Because gene therapy generally involves the delivery of DNA from either plasmids of bacterial origin or recombinant viruses, an acute inflammatory response of variable severity inevitably results. The response is most serious for non-viral gene delivery vectors composed of cationic lipid–DNA complexes, producing adverse effects at lower doses and lethality at higher doses of complex. This review examines the role of immunostimulatory CpG motifs in the acute inflammatory response to non-viral gene therapy vectors. Strategies to neutralise or eliminate CpG motifs within plasmid DNA vectors, and the existing limitations of CpG reduction on improving the safety profile of non-viral vectors, will be discussed.

The hereditary von Hippel-Lindau (VHL) syndrome predisposes sufferers to highly vascularized tumors such as renal clear cell carcinoma (RCC) and central nervous system hemangioblastoma. In RCC4 and RCC786–0 VHL−cells with VHL mutations, the protein of hypoxia-inducible factor-1α (HIF-1α) is constitutively stabilized and the mRNA levels of HIF target genes, including vascular endothelial growth factor (VEGF), are elevated. However, the expression of angiopoietins in these cells and their involvement in angiogenesis are not well known. In this study, we compared the mRNA levels of angiopoietins in human kidney proximal tubule epithelial (RPTE) and RCC4 and RCC786–0 VHL−cells. In RPTE cells, angiopoietin-4 (Ang-4) expression was selectively induced by hypoxia or by expression of a hybrid form of HIF-1α. Under normoxic conditions, the mRNA levels of Ang-4 were higher in RCC4 and RCC786–0 VHL−than RPTE cells. Angiopoietin-1 expression was detectable in RCC4 and RCC786–0 VHL−cells but not RPTE cells. In RCC786–0 VHL+cells, which were stably transfected with a wild-type copy of VHL, the mRNA levels of VEGF and Ang-4 were suppressed and the hypoxic response was restored. We also demonstrated that stimulation of endothelial tube formation by conditioned medium harvested from RCC4 cells was inhibited by a soluble Tie-2 receptor. These results suggest that the angiopoietin/Tie-2 system may participate in the angiogenic response to hypoxia in renal tissues and in tumor angiogenesis in renal carcinoma.

Fas, a member of the tumor necrosis factor receptor super-family, is expressed in all cell types examined, while physiologic expression of Fas ligand (FasL) is found predominantly in activated T-lymphocytes, vascular endothelial cells, and “immune-privileged” tissues. Activation of Fas following FasL binding activates caspases, which results in apoptosis. In the vasculature, there may be a delicate balance between cell proliferation and apoptosis in vascular smooth muscle cells. Shifts in this balance could account for the accumulation of vascular smooth muscle cells in response to arterial injury, a major feature of vascular intimal hyperplasia. Intimal hyperplasia occurs in more than a third of patients receiving percutaneous transluminal balloon angioplasty. Stenting with or without coating significantly reduces the incidence rate of angiographic restenosis and that of target vessel revascularization. However, “in-stent” intimal hyperplasia / restenosis remains a challenge for clinical cardiologists. Although both the cell types and mechanisms that contribute to intimal hyperplasia in response to vascular injury remain controversial, vascular smooth muscle cell migration and proliferation appear to play an important role in the process. In animal models, cytotoxic and cytostatic gene therapy strategies targeted at the vascular smooth muscle cells have shown therapeutic potential for the treatment of vascular intimal hyperplasia. However, Fas ligand-based gene therapy appears to offer several advantages. In this review article, we will discuss the mode of FasL / Fas signaling in vascular smooth muscle cells and its therapeutic implications. We will also compare the relative merits of FasL with other cytotoxic and cytostatic gene therapy approaches for the treatment of intimal hyperplasia.

Enzyme replacement therapy for Gaucher disease uses a recombinant glucocerebrosidase (Cerezyme) whose oligosaccharide chains have been remodeled to expose the core mannose residues. This modification promotes the uptake of the hydrolase by Gaucher-affected macrophages via mannose receptor-mediated endocytosis. However, studies revealed that amounts of the infused enzyme were also delivered to other mannose receptor-bearing cells such as the liver sinusoidal endothelial cells. To maximize the delivery of Cerezyme to macrophages, agents that increased the cell surface levels of the mannose receptor specifically on macrophages were examined. Treatment with dexamethasone improved the in vitro uptake of Cerezyme by a macrophage but not by liver sinusoidal endothelial or hepatocyte cell lines. The enhanced uptake by the macrophages was due to an increase in surface mannose receptors because the activity could be blocked by the addition of mannans. Pretreatment of rats with the glucocorticoid also preferentially enhanced the delivery of Cerezyme to the Kupffer cells and splenic macrophages. This effect of dexamethasone also applied to substrate-laden macrophages isolated from Niemann-Pick A mice. Together, these data suggest that pretreatment with dexamethasone could specifically enhance the presentation of mannose receptors on Gaucher macrophages with resultant improvement in delivery of the enzyme to the affected cells.

The clinical use of doxorubicin, one of the most effective antitumor drugs, is limited by its cardiotoxicity, which results in irreversible cardiomyopathy and congestive heart failure. This study aimed to evaluate a gene therapy approach using adenovirus-mediated expression of p35, a baculoviral antiapoptotic gene, for alleviating doxorubicin-induced cardiomyopathy. In cultured neonatal rat cardiomyocytes, transduction with a recombinant adenoviral vector expressing p35 (Ad2/CMVp35) but not a control adenoviral vector expressing no transgene (Ad2/CMVEV) significantly inhibited doxorubicin-induced increase in cellular reactive oxygen species (ROS), the activity of caspases 8 and 3, cytochrome c release, and apoptosis. Direct injection of Ad2/CMVp35 into the left ventricular wall inhibited myocardial caspase 3 activity and apoptosis and improved left ventricular performance in rats treated with doxorubicin, whereas the same dose of Ad2/CMV β gal encoding β-galactosidase had no effect. These results suggest that adenovirus-mediated expression of p35 protects cardiomyocytes against doxorubicin cardiotoxicity, possibly by inhibiting caspase activity and by reducing cellular ROS levels. Localized delivery of gene transfer vectors expressing an antiapoptotic protein such as p35 to the myocardium may represent a therapeutic approach to alleviate doxorubicin-induced cardiomyopathy.

Systemic delivery of synthetic gene transfer vectors such as cationic lipid:plasmid DNA (pDNA) complexes elicits a range of acute physiologic responses, which in the context of therapeutic gene delivery represent dose-limiting toxicities. The most prominent responses are transient leukopenia, thrombocytopenia, serum transaminase elevations, and elevations of proinflammatory cytokines such as interferon-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α). The unmethylated CpG sequences present in plasmid DNA have been implicated as a major cause of the robust cytokine response that follows systemic administration of cationic lipid:pDNA complexes. However, the factors causing the additional significant toxicities (leukopenia, thrombocytopenia, and serum transaminase elevations) recently shown to be associated with vector administration have not been defined. We show here that DNA sequences, such as immune stimulatory CpG sequences, play a significant role in inducing the additional acute toxicities associated with cationic lipid:pDNA complex administration. Importantly, while methylating these CpG sequences results in greatly reduced cytokine levels, this modification does not eliminate their ability to generate the other systemic toxicities. Examples of non-CpG DNA sequences that induce distinct toxicity profiles when administered systemically in the form of cationic lipid:DNA complexes are also identified. Taken together, these results imply that specific DNA sequences are responsible for a significant portion of the systemic toxicities observed after administration of cationic lipid:pDNA complexes.

Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-αgal encoding human α-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-αgal by 10-fold (from 1011 to 1010 particles/mouse) resulted in a greater than 200-fold loss in transgene expression. In Fabry mice, the reduced expression of α-galactosidase A, using the lower dose of Ad2/CMVHI-αgal, was associated with less than optimal clearance of the accumulated glycosphingolipid (GL-3) from the affected lysosomes. It was determined that this lack of linearity in dose response was not due to an inability to deliver the recombinant viral vectors to the liver but rather to sequestration, at least in part, of the viral vectors by the Kupffer cells. This lack of correlation between dose and expression levels could be obviated by supplementing the low dose of Ad2/CMVHI-αgal with an unrelated adenoviral vector or by depleting the Kupffer cells before administration of Ad2/CMVHI-αgal. Prior removal of the Kupffer cells, using clodronate liposomes, facilitated the use of a 100-fold lower dose of Ad2/CMVHI-αgal (109 particles/mouse) to effect the nearly complete clearance of GL-3 from the affected organs of Fabry mice. These results suggest that practical strategies that minimize the interaction between the recombinant adenoviral vectors and the reticuloendothelial system (RES) may improve the therapeutic window of this vector system. In this regard, we showed that pretreatment of mice with gamma globulins also resulted in significantly enhanced adenovirus-mediated transduction and expression of α-galactosidase A in the liver.

The cellular response to hypoxia depends on rapid posttranslational modifications of proteins as well as regulation of gene expression. We performed serial analysis of gene expression (SAGE) on human cardiac cells under normoxia, subjected to hypoxia, or infected with Ad2/HIF-1α/VP16 (an adenoviral vector expressing a stable hybrid form of hypoxia-inducible factor 1α) or Ad2/CMVEV (an empty vector). Of the 97,646 SAGE tags that were sequenced, 27% matched GenBank entries, while an additional 32% matched expressed sequence tags (ESTs) in UniGene. We analyzed 161 characterized genes or ESTs with a putative identification. Expression of 35, 11, and 46 genes was increased by hypoxia, infection with Ad2/EVCMV, or infection with Ad2/HIF-1α/VP16, respectively, compared with normoxia; conversely, 20, 11, 38 genes, respectively, were expressed at lower levels. Genes regulated by hypoxia were associated with transcription, biosynthesis, extracellular matrix formation, glycolysis, energy production, cell survival, and cell stress. Changes following infection with Ad2/HIF-1α/VP16 mimicked the hypoxic response to a certain extent. Infection with Ad2/CMVEV affected expression of genes that were associated with extracellular matrix formation and membrane trafficking. Differential expression of select genes was confirmed using TaqMan in additional human cardiac cells and rat neonatal ventricular myocytes. These data provide insight into gene expression underlying the diverse and complex cellular response to hypoxia, expression of HIF-1α/VP16, or adenoviral infection.

Adenovirus-mediated gene transfer of Fas ligand (FasL) inhibits neointimal formation in balloon-injured rat carotid arteries. Vascular smooth muscle (VSM) cells coexpressing murine FasL and p35, a baculovirus gene that inhibits caspase activity, are not susceptible to FasL-mediated apoptosis in vitro but are capable of inducing apoptosis of VSM cells that do not express p35. We reasoned that coexpression of p35 in FasL-transduced VSM cells in vivo would promote their survival, enhance FasL-induced apoptosis of adjacent VSM cells, and thereby facilitate a greater inhibition of neointimal formation. In balloon-injured rabbit femoral arteries, either Ad2/FasL/p35 or Ad2/FasL was infused into the injured site and withdrawn 20 min later. Both vectors induced a dose-dependent reduction (p < 0.05) of the neointima-to-media ratio when assessed 14 days later. However, Ad2/FasL/p35 exhibited a significantly greater inhibition of neointimal formation than Ad2/FasL. In a more clinically relevant model of restenosis, rabbit iliac arteries were injured with an angioplasty catheter under fluoroscopic guidance. Adenoviral vectors were delivered locally to the injured site over a period of 2 min, using a porous infusion balloon catheter. Twenty-eight days after gene transfer angiographic and histologic assessments indicated a significant (p < 0.05) inhibition of iliac artery lumen stenosis and neointimal formation by Ad2/FasL/p35 (5 × 1011 particles per artery). The extent of inhibition was comparable to that achieved with Ad2/TK, an adenoviral vector encoding thymidine kinase (5 × 1011 particles per artery) and coadministration of ganciclovir for 7 days. These data suggest that coexpression of p35 in FasL-transduced VSM cells is more potent at inhibiting neointimal formation and as such represents an improved gene therapy approach for restenosis.

Sustained transgene expression will be required for the successful treatment of most genetic diseases being considered for gene therapy. The initially high levels of expression attained with plasmid DNA (pDNA) vectors containing viral promoters, such as that from cytomegalovirus (CMV), decline precipitously to near-background levels within two to three weeks. Here we constructed pDNA vectors containing the human cellular UBB (encoding ubiquitin B; Ub) promoter and evaluated their expression in the mouse lung. Cationic lipid-pDNA complexes were instilled intranasally (IN) or injected intravenously (IV) into immunodeficient BALB/c mice. Chloramphenicol acetyltransferase (CAT) reporter gene expression from the UBB promoter was initially very low at day 2 post-administration, but by day 35 exceeded the level of expression attained from a CMV promoter vector by four- to ninefold. Appending a portion of the CMV enhancer 5′ of the UBB promoter (CMV-Ub) increased CAT expression to nearly that of the CMV promoter and expression persisted in the lung for at least 3 months, with 50% of day 2 levels remaining at day 84. In the liver, expression from the CMV-Ub hybrid promoter was sustained for 42 days. As previous studies have shown that eliminating immunostimulatory CpG motifs in pDNA vectors reduces their toxicity, we constructed a CpG-deficient version of the CMV-Ub vector expressing α-galactosidase A, the enzyme deficient in Fabry disease, a lysosomal storage disorder. After IN or IV administration, levels of α-galactosidase A from this vector were not only undiminished but increased 500% to 1500% by day 35. Our results indicate that CpG-reduced plasmid vectors containing a CMV-Ub hybrid promoter may provide the long-term expression required for a practical gene therapeutic.Molecular Therapy (2001) 4, 75–82; doi: 10.1006/mthe.2001.0415

A major limitation associated with systemic administration of cationic lipid:plasmid DNA (pDNA) complexes is the vector toxicity at the doses necessary to produce therapeutically relevant levels of transgene expression. Systematic evaluation of these toxicities has revealed that mice injected intravenously with cationic lipid:pDNA complexes develop significant, dose-dependent hematologic and serologic changes typified by profound leukopenia, thrombocytopenia, and elevated levels of serum transaminases indicative of hepatocellular necrosis. Vector administration also induced a potent inflammatory response characterized by complement activation and the induction of the cytokines IFN-γ, TNF-α, IL-6, and IL-12. These toxicities were found to be transient, resolving with different kinetics to pretreatment levels by 14 days posttreatment. The toxic syndrome observed was independent of the cationic lipid:pDNA ratio, the cationic lipid species, and the level of transgene expression attained. Mechanistic studies determined that neither the complement cascade nor TNF-α were key mediators in the development of these characteristic toxicities. Administration of equivalent doses of the individual vector components revealed that cationic liposomes or pDNA alone did not generate the toxic responses observed with cationic lipid:pDNA complexes. Only moderate leukopenia was associated with administration of cationic liposomes or pDNA alone, while only mild thrombocytopenia was noted in pDNA-treated animals. These results establish a panel of objective parameters that can be used to quantify the acute toxicities resulting from systemic administration of cationic lipid:pDNA complexes, which in turn provides a means to compare the therapeutic indices of these vectors.

An inflammatory response is invariably associated with administration of gene transfer complexes composed of cationic lipids and plasmid DNA (pDNA). In the lung, an influx of neutrophils and elevated levels of several proinflammatory cytokines such as TNF-α, IFN-γ, IL-6, and IL-12 characterize this dose-dependent response. The induction of these cytokines was shown previously to be due in part to the presence of unmethylated CpG dinucleotides in the bacterially derived pDNA. We have eliminated 270 of 526 CpG dinucleotides in a reporter plasmid (pCFA-CAT) and tested the inflammatory response to cationic lipid:pDNA complexes containing the modified vector (pGZA-CAT) after intravenous (iv) or intranasal (i.n.) delivery into BALB/c mice. Compared to the unmodified vector, the CpG-reduced pGZA-CAT was found to be significantly less immunostimulatory, as the levels of IL-12, IFN-γ, and IL-6 in the serum 24 h after iv delivery were reduced by 40 to 75%. Similar reductions in cytokine levels were also observed in the bronchoalveolar lavage fluids (BALF) after i.n. administration, while the levels of reporter gene expression were not affected by the modifications. We have also investigated known inhibitors of the CpG signaling pathways in order to decrease the inflammatory response. Two such inhibitors, chloroquine and quinacrine, greatly reduced the induction of IL-12 from mouse spleen cells in vitro and inhibited cytokine production in the lung by approximately 50% without affecting gene expression. These results illustrate that use of a less immunostimulatory pDNA vector or inhibitors of CpG immunostimulation can reduce significantly the toxicity associated with cationic lipid:pDNA complexes thereby increasing the therapeutic index of this synthetic gene transfer vector.Molecular Therapy (2000) 1, 255–262; doi: 10.1006/mthe.2000.0036

AbstractEnhanced gene transduction to the lung using cationic lipids could be attained through optimization of the structure of the lipids and the formulation of the cationic lipid : plasmid DN A (pDNA) complexes. We have expanded on our earlier observation of the importance of the structural orientation of the cationic lipid headgroup. Through the synthesis of a number of matched pairs of cationic lipids differing only in the configuration of their headgroup, we confirmed that those harboring a T-shape headgroup are more active than their linear counterparts, at least when tested in the lungs of BALB/c mice. Additionally, we demonstrated that not only are the structural considerations of these cationic lipids important, but also their protonation state, the free base being invariably more active than its salt counterpart. The salt forms of cationic lipids bound pDNA with greater avidity, which may have affected their subsequent intracellular dissolution and transit of the pDNA to the nucleus. Inclusion of a number of frequently used solutes in the vehicle severely inhibited the gene transfection activity of the cationic lipids. The selection of neutral co-lipids was also an important factor for overall transfection activity of the formulation, with significant gains in transfection activity realized when diphytanoylphosphatidylethanol-amine or dilinoleoylphosphatidylethanolamine were used in lieu of dioleoylphosphati-dylethanolamine. Finally, we showed that a transacylation reaction could occur between the cationic lipid and neutral co-lipid which reduced the transfection activity of the complexes. It is the hope that as our understanding of the many factors that influence the activity of these cationic lipid: pDNA complexes improves, formulations with much greater potency can be realized for use in the treatment of pulmonary diseases.

For gene therapy to be effective in the treatment of chronic diseases, plasmid DNA (pDNA) vectors that provide persistent expression of therapeutic levels of the transgene product are desirable. Studies in the lung with adenovirus vectors showed that products of the adenovirus E4 region can act both in cis and in trans to increase the duration of expression when transcription of the transgene was under the control of the human cytomegalovirus (CMV) promoter. To determine if these E4-encoded proteins could also effect greater persistence of expression from a nonviral vector, a complex composed of cationic lipid GL-67, a CMV promoter plasmid (pCF1-CAT), and an E4-containing adenovirus vector (Ad2/beta gal-4) was instilled into the lungs of BALB/c nu/nu mice. Significant increases in the duration of transgene expression were observed for up to 10 weeks postinstillation compared with expression from mice instilled with control complexes containing an adenovirus vector deleted of most of E4 (Ad2/beta gal-2). This effect could also be observed in immunodeficient NIH-rnu rats as well as in immunocompetent BALB/c mice. Studies with CMV promoter mutants indicated that a region proximal to the promoter was necessary for the E4-mediated increase in longevity of expression. In addition to the CMV promoter, a CMV enhancer-human mucin I (MUC-I) hybrid promoter also responded to these E4-encoded proteins with increased persistence of transgene expression, but a human interleukin 8 (IL-8) promoter did not. Ad2/beta gal-4 could be replaced by a pDNA vector expressing only the E4 region, indicating that products of the E4 region alone were sufficient in the absence of expression from the rest of the adenovirus genome. Further analysis indicated that the protein encoded by open reading frame 3 (ORF3) alone was sufficient for conferring the increase in persistence of expression. These data indicate that expression of a single protein from the adenovirus genome can significantly improve the duration of transgene expression from pDNA vectors, and increases the feasibility of using nonviral vectors for the treatment of chronic diseases.

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEH alpha-Gal, Ad2/CMVHI alpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEH alpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. alpha-Galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with Sss I-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible causeand-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1 alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.

Here we describe the production of cystic fibrosis transmembrane conductance regulator (CFTR), the product of the gene associated with cystic fibrosis, in the milk of transgenic mice. Mammary specific expression was achieved by placing the CFTR cDNA under the control of the goat β-casein gene promoter. By fractionation, CFTR was shown to be associated with the membranes that envelop milk fat globules as they are discharged from the apical surface of the mammary epithelia. Since milk fat globules may comprise up to 10% of whole milk, this represents a novel, inexpensive and efficient approach to produce CFTR and possibly other membrane-associated proteins. The availability of large quantities of CFTR could have important implications for the development of new therapies for cystic fibrosis.

Gene delivery with cationic liposomesformed from the diamine‐lipid DC‐Chol and the neutral lipid DOPE has proven successful. New polyamine analogues of DC‐Chol have been synthesised, formulated into cationic liposomes with DOPE and biologically tested in vitro and in vivo in mouse lung. Cationic liposomes containing the novel DC‐Chol analogue CTAP (shown below) work 100 times more effectively in vivo than DC‐Chol containing liposomes (500 times better than DNA alone) and therefore should be suitable for use in human gene therapy approaches towards lung disorders and other clinical conditions.

ABSTRACTSystematic analysis of a large number of different cationic lipids has led to the identification of novel structures (GL-67) and formulations of cationic lipid:plasmid DNA (pDNA) complexes that facilitate high levels of gene expression in lungs of mice. However, despite significant improvement in gene transfer activity, we show here that the efficiency of GL-67-mediated gene transduction of intact airway epithelia is still relatively low. Administration of GL-67:pCF1-CFTR (encoding the cystic fibrosis transmembrane conductance regulator) complexes into the nasal epithelium of cystic fibrosis (CF) transgenic mice resulted only in marginal correction of the ion transport defects. Measurements of nasal potential differences (PD) showed no correction of the sodium (Na) transport defect, and only partial restitution of the chloride (Cl) transport defect was achieved in a small proportion of the animals after perfusion of the nasal epithelium with the complexes. Furthermore, in contrast to results obtained following instillation of GL-67:pDNA complexes into the lungs of mice, perfusion of GL-67:pDNA into the nasal epithelium resulted only in a moderate enhancement of gene transduction activity relative to that attained with naked pDNA alone. To determine the basis for this low efficiency of transfection, a series of studies was conducted to identify some of the barriers governing cationic lipid-mediated gene transfer to the airway epithelium. We show here that the transfection activity of GL-67 was affected by the polarization, differentiation, and proliferative state of the cells. Diminished transfection activity was observed with nonmitotic, highly polarized and differentiated airway epithelial cells. This observed reduction in gene expression with nonmitotic cells was determined to be due in part to inefficient nuclear translocation of the pDNA from the cytoplasm. Together these data indicate that much improvement in the ability of cationic lipids to transfect polarized and differentiated airway epithelial cells is a necessary prerequisite for effective cationic lipid-mediated gene therapy of airway diseases such as CF.

ABSTRACTRecombinant adenovirus (Ad) vectors are being considered for in vivodelivery of various therapeutic genes. One limiting factor in the development of Ad-based gene therapy is the low efficiency of gene transfer to target tissues such as vascular endothelium, smooth muscle, and airway epithelium. Complexing Ad vector with various polycations has been shown to enhance transduction of cell lines otherwise resistant to Ad infection in vitro. On the basis of this observation, the activity of Ad/polycation complexes was tested in vivoin the mouse lung. The results indicated that several polycations were capable of enhancing transduction of mouse respiratory epithelium, leading to a 12 log increase in levels of transgene expression. Poly-l-lysine (PLL) and DEAE-dextran were examined further and were found to increase Ad-mediated gene transfer without any additional toxicity as assessed histologically or through the measurement of inflammatory cytokines in bronchoalveolar lavages. The two polycations also failed to affect the humoral response against Ad vector and were themselves nonimmunogenic under conditions leading to enhanced gene transfer. Moreover, the ability to use reduced doses of vector complexed with polycations resulted in lower levels of Ad-specific antibodies and, thereby, improved readministration of vector. These results suggest that complexing Ad vectors with polycations has the potential to improve the therapeutic index by increasing transgene expression while reducing unwanted responses associated with high doses of vector.

ABSTRACTCationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a T-shape configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 g of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTRgene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTRin CF lungs is a viable and promising approach for treatment of the disease.

We have generated mice carrying the most common mutation in cystic fibrosis (CF), ΔF508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. ΔF508−/−mice die from peritonitis and show deficiencies in cAMP–activated electrogenic Cl−transport. These mice produce ΔF508 transcripts and show the temperature–dependent trafficking defect first described for the human ΔF508 CFTR protein. A functional CFTR Cl−channel not demonstrated by null CF mice or present at 37 °C was detected following incubation of epithelial cells at 27 °C. Thus, these mice are an accurate ΔF508 model and will be valuable for testing drugs aimed at overcoming the ΔF508 trafficking defect.

Gene delivery with cationic liposomes formed from the diamine-lipid DC-Chol and the neutral lipid DOPE has proven successful. New polyamine analogues of DC-Chol have been synthesised, formulated into cationic liposomes with DOPE and biologically tested in vitro and in vivo in mouse lung. Cationic liposomes containing the novel DC-Chol analogue CTAP (shown below) work 100 times more effectively in vivo than DC-Chol containing liposomes (500 times better than DNA alone) and therefore should be suitable for use in human gene therapy approaches towards lung disorders and other clinical conditions.

ABSTRACTPreviously, we have described the optimization of the aerosol delivery of a nonviral gene therapy vector to the lungs of rodents (Eastman et al., 1997b). Although aerosolizing cationic lipid:pDNA complexes into a whole-body exposure chamber resulted in high levels of reporter gene expression in the lungs of BALB/c mice, the conditions employed were not optimal for the delivery of lipid:pDNA complexes to the lungs of human patients. That is, the consumption rate of the material in the nebulizer, and thus the delivery time, were very slow and the aerosol was delivered in a continuous flow. Here we describe in vitroexperiments used to develop a cationic lipid:pDNA aerosol with characteristics more suitable for delivery to the lungs of humans, as a necessary prerequisite for conducting a clinical study with human cystic fibrosis patients. Using cascade impactors and all-glass impingers, we have screened several commercially available nebulizers for their ability to deliver intact, respirable, active lipid:pDNA complexes in the shortest time possible, and have identified the Pari LC Jet Plus nebulizer as the optimal nebulizer that meets these criteria. Using this nebulizer in an intermittent mode to mimic breath actuation, consumption rates of approximately 0.6 ml/min of the cationic lipid:pDNA complexes (6 mMcationic lipid:8 mMpDNA) were obtained. The plasmid DNA remained intact and the complexes were shown to maintain activity throughout the nebulization run. Based on measurements of the nebulized dose and the mass median aerodynamic diameter, we calculate a delivered dose of ~ 22 mol (7.2 mg) of pDNA for each 8 ml of cationic lipid:pDNA complex aerosolized to the lungs of a human patient. This dose should be sufficient to test the clinical efficacy of cationic lipid-mediated gene delivery for the treatment of cystic fibrosis.

ABSTRACTWe have examined several variables inherent in aerosolizing cationic lipid:DNA complexes using a jet nebulizer and thereby have optimized the delivery of functional complexes. Maximal aerosol transfer efficiency of cationic lipid:pDNA complexes was quantitated and shown to require the presence of at least 25 mMNaCl as an excipient. This is possibly related to effects on the measured zeta potentials of the complex, which indicate that the complexes are more highly charged in solutions of physiological ionic strength than in solutions of low ionic strength. Inclusion of saline also resulted in retention of the starting lipid to plasmid DNA (pDNA) ratio following nebulization. These data were used to design in vitroaerosolization experiments with tissue culture cells that resulted in the identification of a cationic lipid:pDNA ratio of 0.75:1 (mol:mol) as being optimal for aerosolization. This formulation largely protected pDNA from shear degradation during nebulization and produced a respirable aerosol droplet size (13 m). It was tested further in a mouse model and shown to result in the dose-dependent transfection of mouse lungs, generating the equivalent of several picograms of reporter gene activity per mouse lung. The results of these experiments have provided a set of optimal conditions for nebulizing cationic lipid:pDNA complexes that can be used as a starting point for the further evaluation of aerosol delivery of these nonviral gene delivery vectors in vivo.

ABSTRACTAdvances in gene therapy vectors and techniques hold promise for treatment of many inherited and acquired diseases. For lung indications, especially those involving the epithelium, delivery of the gene therapy vehicle ideally will involve the use of an aerosol. Aerosol delivery of transgenes using cationic lipids is currently limited by the ability to generate highly concentrated formulations of lipid:DNA complexes that are stable and retain their activity following aerosolization. We have examined many of the variables inherent in aerosolizing cationic lipid gene delivery vehicles and have devised a new formulation that incorporates small amounts of a polyethylene glycol-containing lipid. This formulation has allowed the preparation of concentrated dispersions of cationic lipid:plasmid DNA (pDNA) complexes (>20 mMpDNA) at approximately 10-fold higher concentrations than previously reported. Most of the pDNA in these formulations was bound to the lipid component and thereby protected from nebulizer-induced shearing; the pDNA also maintained full biological activity both in vitroand in vivo. This new formulation thus represents a significant improvement over current methods to prepare concentrated, active cationic lipid gene delivery vectors, and provides a new tool with which to test gene transfer to the lung.

ABSTRACTMultiple dosing with recombinant adenoviral vectors containing the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the nasal mucosa of cystic fibrosis (CF) transgenic mice reportedly results in only partial correction of the CF defect in chloride (Cl¯¯) secretion without normalizing sodium (Na+) hyperabsorption, perhaps indicating inefficient gene transfer into the nasal airway epithelium in vivo. In this study, we have examined whether optimizing vector administration such as contact time could improve gene transfer efficiency. Changes in basal nasal potential difference (PD), and in PD (ΔΔPD) following addition of amiloride and subsequent removal of Cl¯¯ from the luminal perfusate were assayed. As reported previously, the basal nasal PD was significantly more negative in CF mice (––24.9 ±± 2.1 mV) than in normal mice (––6.3 ±± 1.2 mV). Normal mouse nasal mucosa exhibited a large hyperpolarization in response to low Cl¯¯ substitution (ΔΔPD of 8.5 ±± 1.9 mV), whereas the nasal mucosa of the CF mouse depolarized in response to this treatment. No correction of either the Cl¯¯ or Na+transport defects were observed when 5 ×× 109IU of Ad2/CFTR-5 were administered to the nasal passage of CF mice over a period of 5––20 min. However, when CF mice were perfused over a period of 60 min with the same dose of vector, a significant response (ΔΔPD of 5.9 ±± 1.1 mV) to low Cl¯¯ substitution was detected 2 days later. In these mice, the basal nasal PD (––10.5 ±± 1.4 mV) and the response to amiloride were also reduced, indicating a partial correction of the Na+transport defect. Expression of functional CFTR activity was transient with no measurable ΔΔPD signals observed by day 7 post-treatment. These results suggest that prolonging the contact between an adenoviral vector and the respiratory epithelium enhances the efficiency of gene transfer and can result in improved correction of the CF Na+and Cl¯¯ ion transport defects. Therefore, strategies that improve internalization of viral vectors and that prolong their contact time with target cells may result in the improved clinical efficacy of such vectors.