AAV8(Y733F)-mediated gene therapy in a Spata7 knockout mouse model of Leber congenital amaurosis and retinitis pigmentosa.

Loss of SPATA7 function causes the pathogenesis of Leber congenital amaurosis and retinitis pigmentosa. Spata7 knockout mice mimic human SPATA7-related retinal disease with apparent photoreceptor degeneration observed as early as postnatal day 15 (P15). To test the efficacy of adeno-associated virus (AAV)-mediated gene therapy for rescue of photoreceptor survival and function in Spata7 mutant mice, we employed the AAV8(Y733F) vector carrying hGRK1-driven full-length FLAG-tagged Spata7 cDNA to target both rod and cone photoreceptors. Following subretinal injection of this vector, FLAG-tagged SPATA7 was found to colocalize with endogenous SPATA7 in wild-type mice. In Spata7 mutant mice initially treated at P15, we observed improvement of photoresponse, photoreceptor ultrastructure and significant alleviation of photoreceptor degeneration. Furthermore, we performed treatments at P28 and P56 and found that all treatments (P15-P56) can ameliorate rod and cone loss in the long term (1 year); however, none efficiently protect photoreceptors from degeneration by 86 weeks of age as only a small amount of treated photoreceptors can survive to this time. This study demonstrates long-term improvement of photoreceptor function by AAV8(Y733F)-introduced Spata7 expression in a mouse model as potential treatment of the human disease, but also suggests that treated mutant photoreceptors still undergo progressive degeneration.

Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus.

Usher 1 patients are born profoundly deaf and then develop retinal degeneration. Thus they are readily identified before the onset of retinal degeneration, making gene therapy a viable strategy to prevent their blindness. Here, we have investigated the use of adeno-associated viruses (AAVs) for the delivery of the Usher 1B gene, MYO7A, to retinal cells in cell culture and in Myo7a-null mice. MYO7A cDNA, under control of a smCBA promoter, was packaged in single AAV2 and AAV5 vectors and as two overlapping halves in dual AAV2 vectors. The 7.9-kb smCBA-MYO7A exceeds the capacity of an AAV vector; packaging of such oversized constructs into single AAV vectors may involve fragmentation of the gene. Nevertheless, the AAV2 and AAV5 single vector preparations successfully transduced photoreceptor and retinal pigment epithelium cells, resulting in functional, full-length MYO7A protein and correction of mutant phenotypes, suggesting successful homologous recombination of gene fragments. With discrete, conventional-sized dual AAV2 vectors, full-length MYO7A was detected, but the level of protein expression was variable, and only a minority of cells showed phenotype correction. Our results show that MYO7A therapy with AAV2 or AAV5 single vectors is efficacious; however, the dual AAV2 approach proved to be less effective.

rAAV2/5 gene-targeting to rods:dose-dependent efficiency and complications associated with different promoters.

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken ß actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 10(11)vg ml(-1) were used.

AAV retinal transduction in a large animal model species: comparison of a self-complementary AAV2/5 with a single-stranded AAV2/5 vector.

PURPOSE: To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection. METHODS: ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity. RESULTS: GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced. CONCLUSIONS: When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.

Targeting gene expression to cones with human cone opsin promoters in recombinant AAV.

Specific cone-directed therapy is of high priority in the treatment of human hereditary retinal diseases. However, not much information exists about the specific targeting of photoreceptor subclasses. Three versions of the human red cone opsin promoter (PR0.5, 3LCR-PR0.5 and PR2.1), and the human blue cone opsin promoter HB569, were evaluated for their specificity and robustness in targeting green fluorescent protein (GFP) gene expression to subclasses of cones in the canine retina when used in recombinant adeno-associated viral vectors of serotype 5. The vectors were administered by subretinal injection. The promoter PR2.1 led to most effective and specific expression of GFP in the long- and medium-wavelength-absorbing cones (L/M cones) of normal and diseased retinas. The PR0.5 promoter was not effective. Adding three copies of the 35-bp LCR in front of PR0.5 lead to weak GFP expression in L/M cones. The HB569 promoter was not specific, and GFP was expressed in a few L/M cones, some rods and the retinal pigment epithelium. These results suggest that L/M cones, the predominant class of cone photoreceptors in the retinas of dogs and most mammalian species can be successfully targeted using the human red cone opsin promoter.

Modulation of the expression of the transcription factor Max in rat retinal ganglion cells by a recombinant adeno-associated viral vector.

Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.

Genetic and DNA sequence analysis of the Salmonella typhimurium virulence plasmid gene encoding the 28,000-molecular-weight protein.

We have confirmed that the 28,000-molecular-weight (28K) protein encoded by the virA gene of the 90-kilobase Salmonella typhimurium virulence plasmid is a virulence factor. It was previously shown that a Tn5 insertion, vir-22::Tn5, located in the virulence plasmid greatly attenuated virulence for mice and inhibited the production of a 28K protein (P.A. Gulig and R. Curtiss III, Infect. Immun. 56:3262-3271, 1988). Plasmid pYA426 fully complemented vir-22::Tn5 to virulence by increasing splenic infection after oral inoculation and encoded the 28K protein. To identify the virulence gene(s) of pYA426 mutated by vir-22::Tn5, we constructed nested deletions in pYA426 and examined deletion derivatives for their abilities to complement vir-22::Tn5. Only derivatives still producing the 28K protein complemented vir-22::Tn5. Furthermore, the smallest complementing derivative encoded only the 28K protein, as determined by DNA sequence analysis. Therefore, the 28K protein is sufficient for complementation of the attenuating mutation vir-22::Tn5 and must be the virulence factor inhibited by the insertion. We determined the nucleotide sequence of the 1.2-kilobase BamHI-EcoRI fragment encoding the 28K protein and identified the structural gene, virA. A 723-base-pair open reading frame which encodes a peptide with a molecular weight of 27,572 was found.

Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid.

The 90-kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14-kb virulence region to reduce this virulence region to 7.8kb. The 7.8-kb virulence region subcloned into a low copy-number vector conferred a wild-type level of splenic infection to virulence plasmid-cured S. typhimurium and conferred essentially a wild-type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild-type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho-dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8-kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid-encoded virulence genes of S. typhimurium.

Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo.

The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.

Age-dependent antibody response in mice and humans following oral influenza immunization.

In order to compare the antibody response in serum and secretions from healthy young subjects and the elderly (greater than 60 years), volunteers were immunized with the commercial inactivated influenza virus vaccine, by the usual (parenteral) route or orally. Also, young and old mice (mean age, 20 months) were orally immunized with live influenza virus. The older mice responded with a very slight rise in their serum and respiratory tract antibody levels compared with the young mice but showed no diminution in protection against lethal viral challenge. Elderly volunteers showed only slight serum antibody responses after parenteral immunization compared with the young. Neither group demonstrated a rise in serum antibody following oral immunization. With respect to the secretory IgA (SIgA) antibody response, certain differences were noted between the young and the elderly: the preimmunization levels of antibody to influenza virus were significantly greater in nasal secretions and saliva in the elderly as compared to the young volunteers, and the salivary antibody response was diminished in the elderly. This lack of a salivary antibody response in the elderly was explicable by the inverse relationship between the preimmunization SIgA antibody titers and the response to immunization. Oral immunization led to no more side effects than observed in the placebo control group.

Modulation of the expression of the transcription factor Max in rat retinal ganglion cells by a recombinant adeno-associated viral vector

Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.