Novel AAV serotypes for improved ocular gene transfer

Some of the most successful gene therapy results have been obtained using recombinant viral vectors to treat animal models of inherited and acquired ocular diseases. Clinical trials using adenovirus vector systems have been initiated for two ocular diseases. Adeno-associated viruses (AAVs) represent an attractive alternative to adenoviral vector systems as they enable stable and long-term expression and can target a variety of different ocular cell types depending on the capsid serotype; recently clinical trails for congenital blindness was initiated with a vector-based AAV serotype 2. High levels of retinal gene transfer have been achieved using vectors based on AAV serotypes 1, 2, 4 and 5. This report compares the gene transfer efficacy and stability of expression of vector systems based on three novel AAV serotypes: AAV7, 8, 9, with the established vectors AAV1, 2, 5. We show here that AAV7 and 8 enable superior long-term transduction of retinal and also anterior chamber structures.     

Improving AAV vector yield in insect cells by modulating the temperature after infection

Vectors based on adeno-associated viruses (AAV) are sought for therapeutic gene delivery because of their ability to transduce a variety of tissues with no significant immunological response. Production using the baculovirus expression vector (BEV)/insect cell system has the potential to meet the needs for pre-clinical and clinical trials. In this co-infection system, three baculoviruses are used to produce the AAV vector. A strategy aimed at increasing encapsidation/maturation of the viral vector involved varying the temperature over the course of the process. Cultures were subjected to temperature changes at various times pre- and post-infection (up to 24 h post-infection). It was found that raising the culture temperature to 30 degrees C at the time of infection nearly tripled the infectious titer. In fact, increasing the temperature to 30 degrees C at any time in the process investigated resulted in an increase in titer. Also, raising the culture to 33 degrees C or lowering the temperature to 24 degrees or 21 degrees C resulted in lower titers. The rise in infectious titer was also confirmed by an increase in DNase resistant particles (DRPs). Varying the temperature, however, did not affect the total amount of capsids significantly. Therefore increasing the culture temperature resulted in better encapsidation as determined by the ratio of capsids to DRPs to infectious particles. It is believed that an increase in early proteins and possibly a quicker cascade of baculovirus infection events resulted in this increased packaging efficiency.     

Femoral intra-arterial injection: a tool to deliver and assess recombinant AAV constructs in rodents whole hind limb

With the aim of simplifying recombinant-adeno-associated virus (rAAV) delivery in muscle, a new femoral intra-arterial technique was designed and tested in rodents (rats and mice). Two serotypes, several promoters and transgenes (reporter or therapeutic) were tested using this administration route. The new route is both easy to perform and efficient. Its usefulness as a tool to assess gene delivery constructs in the muscle was established in the context of recombinant AAV serotypes 1 and 2, and with the ubiquitous CMV and two muscle-specific (C5-12 and CK6) promoters. Both serum monitoring of a secreted protein (murine alkaline phosphatase: muSEAP) and slide staining were used to compare the different constructs. Significantly different patterns of expression in kinetics of expression (muSEAP) and homogeneity of fiber transduction (staining) were evidenced with the different promoters tested, and compared with intra-muscular expression patterns. Detailed studies of differential transduction in leg and thigh muscles showed equivalent efficacy, except in rectus femoris, and to a lesser extent in soleus. In light of these results and prior data, intra-arterially mediated gene transfer mechanism is discussed.     

The transient expression of mRNA coding for Rep protein from AAV facilitates targeted plasmid integration

BACKGROUND: There is a risk of insertional mutagenesis when techniques that facilitate random integration of exogenous DNA into the human genome are used for gene therapy. Wild-type adeno-associated virus (AAV) integrates preferentially into a specific site on human chromosome 19 (AAVS1). This is mediated by the interaction of the viral Rep68/78 proteins with Rep-binding elements in the AAV genome and AAVS1. This specificity is often lost when AAV is used as a gene therapy vector due to removal of the sequences coding for Rep. METHODS: Messenger RNA coding for the Rep68/78 proteins was prepared in vitro and co-transfected with a 21 kb DNA plasmid containing the P5 integration efficiency element (P5IEE) from AAV. Single cells were seeded in plates to establish clonal cell lines that were subsequently analysed by dual colour fluorescent in situ hybridisation (FISH) to determine whether site-specific plasmid integration had occurred on chromosome 19. RESULTS: The co-transfection of plasmid DNA with Rep68/78 mRNA gave a 2.5-fold increase in DNA integration when compared to transfection of cells with plasmid DNA alone. Rep68/78 mRNA expression facilitated site-specific plasmid integration to chromosome 19 in 30% (14/44) of all analysed integration sites, while no targeted integration events were observed following transfection of cells with plasmid DNA alone. CONCLUSIONS: These results demonstrate that transient expression of Rep protein using transfected mRNA facilitates site-specific integration of plasmid DNA. This approach allows expression of Rep for only a short time, and may circumvent the toxicity and chromosome instability associated with long-term expression of Rep.     

Sustained delivery of therapeutic concentrations of human clotting factor IX--a comparison of adenoviral and AAV vectors administered in utero

Background

Prenatal somatic gene therapy has been considered for genetic disorders presenting with morbidity at birth. Haemophilia is associated with an increased risk of catastrophic perinatal bleeding complications such as intracranial haemorrhage, which could be prevented by gene transfer in utero. Prenatal gene therapy may be more promising than postnatal treatment, as the fetus may be more amenable to uptake and integration of therapeutic DNA and the immaturity of its immune system may permit life‐long immune tolerance of the transgenic protein, thus avoiding the dominant problem in haemophilia treatment, the formation of inhibitory antibodies.

Methods

Adenovirus serotype 5‐derived or AAV serotype 2‐derived vectors carrying human clotting factor IX (hfIX) cDNA or a reporter gene were administered intramuscularly, intraperitoneally or intravascularly to late‐gestation mouse fetuses. Both vector types were evaluated with respect to the kinetics of hfIX delivery to the systemic circulation and possible immune responses against the vector or the transgene product.

Results

Mice treated in utero by intramuscular injection of an adenoviral vector carrying hfIX cDNA exhibited high‐level gene expression at birth and therapeutic – albeit continuously decreasing – plasma concentrations of hfIX over the entire 6 months of the study. Adenoviral vector spread to multiple organs was detected by polymerase chain reaction (PCR). Intramuscular, intraperitoneal or intravascular application of AAV vectors carrying hfIX cDNA led to much lower plasma concentrations of hfIX shortly after birth, which appeared to decline during the first month of life but stabilized in some of the mice at detectable levels. No signs of immune responses were found, either against the different viral vectors or against hfIX.

Conclusion

This study demonstrates for the first time that sustained systemic delivery of a therapeutic protein can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy in utero and provides a basis for considering this concept as a preventive therapeutic strategy for haemophilia and perhaps also for other plasma protein deficiencies. Copyright © 2002 John Wiley & Sons, Ltd.

     

Gene transfer into skeletal muscle using novel AAV serotypes

BACKGROUND: Skeletal muscle is an interesting target for gene delivery because of its mass and because the vectors can be delivered in a noninvasive way. Adeno-associated virus (AAV) vectors are capable of transducing skeletal muscle fibers and achieving stable and safe transgene expression. To date, most animal experiments using AAV have been based on AAV serotype 2, but some recent studies have demonstrated that AAV1 is more efficient than AAV2/2 in transducing muscle fibers. Recently, novel AAVs (AAV7 and AAV8) were isolated from rhesus macaques. METHODS: We injected three different muscles (gastrocnemius, soleus, biceps femoris) of immunocompetent C57BL/6 mice with different pseudotyped AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7 and AAV2/8) and quantitatively compared the different gene transfer efficiencies. RESULTS: The efficiencies of transduction in skeletal muscle with AAV2/7 and AAV2/8 were similar to AAV2/1, and higher than that seen with AAV2/2 and AAV2/5. All serotypes were able to transduce both slow and fast muscle fibers similarly at the vector titer used (1x10(11) genome copies per mouse). Despite a limited inflammatory response (slightly higher when using AAV2/2, AAV2/7 and AAV2/8 vectors than AAV2/1 and AAV2/5), transgene expression was observed throughout the length of the experiment. DISCUSSION: These results show that AAV2/7 and AAV2/8 are able to transduce muscle fibers of immunocompetent mice very efficiently, offering new perspectives in gene transfer of skeletal muscle.     

Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle

BACKGROUND: The overlapping approach was developed recently to expand the adeno-associated viral (AAV) packaging capacity. In this approach, a gene is split into two partially overlapping fragments and separately packaged into an upstream and a downstream vector, respectively. Transgene expression is achieved in co-infected cells after homologous recombination. Despite the promising proof-of-principle results in the lung, the efficiency has been very disappointing in skeletal muscle. Here we examined two potential rate-limiting factors including AAV serotype and the transgene sequence. METHODS: To study serotype effect, we delivered AAV-2, -5 and -6 overlapping vectors (5 x 10(8) vg particles of the upstream and the downstream vectors, respectively) and 5 x 10(8) vg particles of the intact gene vector to the tibialis anterior muscles of 7-week-old C57Bl/6 mice, respectively. To determine the effect of transgene sequence, we compared LacZ and alkaline phosphatase (AP) overlapping vectors. Transduction efficiency was quantified 6 weeks later by scoring the percentage of transgene-positive myofibers. RESULTS: AAV-2 overlapping vectors barely resulted in detectable transduction. Transduction efficiency was significantly improved in AAV-5 and AAV-6. The highest level was achieved in AAV-6 that reached 42% and 96% of that of the intact gene vector for the LacZ gene and the AP gene, respectively. Surprisingly, AAV-6 overlapping vector resulted in higher transduction than did AAV-2 and AAV-5 intact gene vectors. CONCLUSIONS: Our findings suggest that AAV serotype and the transgene sequence play critical roles in the overlapping approach. AAV-6 holds great promise for overlapping vector-mediated muscle gene therapy.     

Efficient adeno-associated virus-mediated gene expression in human placenta-derived mesenchymal cells

Mesenchymal cells from various sources are pluripotent and are attractive sources for cell transplantation. In this study, we analyzed recombinant adeno-associated virus (rAAV)-mediated gene expression in human placenta-derived mesenchymal cells (hPDMCs), which reside in placental villi. After transduction of AV-CAG-EGFP, a rAAV expressing enhanced green fluorescence protein (EGFP), hPDMCs showed much higher level of EGFP expression than human umbilical vein endothelial cells or rat aortic smooth muscle cells. The number of EGFP-positive hPDMCs infected by AV-CAG-EGFP alone did not increase significantly by coinfection of adenovirus, which enhanced expression level of the rAAV vector. Moreover, flow cytometric analysis showed discrete positive fraction of EGFP-expressing hPDMCs, which is about 15-20% of the cells infected with AV-CAG-EGFP. Therefore, some cell population in hPDMCs might be highly susceptible to rAAV-mediated gene transduction. In addition, stable EGFP expressions were observed in about 1% of hPDMCs infected with AV-CAG-EGFP at 4 weeks post-infection. Collectively, hPDMCs have characters favorable for rAAV-mediated gene expression.     

Gene therapy for achromatopsia

The present review summarizes the current status of achromatopsia (ACHM) gene therapy‐related research activities and provides an outlook for their clinical application. ACHM is an inherited eye disease characterized by a congenital absence of cone photoreceptor function. As a consequence, ACHM is associated with strongly impaired daylight vision, photophobia, nystagmus and a lack of color discrimination. Currently, six genes have been linked to ACHM. Up to 80% of the patients carry mutations in the genes CNGA3 and CNGB3 encoding the two subunits of the cone cyclic nucleotide‐gated channel. Various animal models of the disease have been established and their characterization has helped to increase our understanding of the pathophysiology associated with ACHM. With the advent of adeno‐associated virus vectors as valuable gene delivery tools for retinal photoreceptors, a number of promising gene supplementation therapy programs have been initiated. In recent years, huge progress has been made towards bringing a curative treatment for ACHM into clinics. The first clinical trials are ongoing or will be launched soon and are expected to contribute important data on the safety and efficacy of ACHM gene supplementation therapy.