Gregory E. Tullis

Characterization of the Transcription Profile of Adeno-Associated Virus Type 5 Reveals a Number of Unique Features Compared to Previously Characterized Adeno-Associated Viruses

ABSTRACT We report the initial characterization of adeno-associated virus type 5 (AAV5) RNAs generated following viral infection and the construction of a replicating infectious clone of AAV5. While the basic transcription profile of AAV5 was similar to that of AAV2, there were also significant differences. Mapping of the AAV5 transcripts demonstrated an efficient transcription initiation site within the AAV5 inverted terminal repeat (ITR), and mapping of the AAV5 intron revealed that it is considerably smaller than that of AAV2. Furthermore, in contrast to the case for AAV2, neither the Rep protein nor additional adenovirus gene products were required to achieve efficient promoter activity and pre-mRNA splicing following transfection of an AAV5 rep/cap plasmid clone lacking the ITRs into 293 cells. Perhaps most surprisingly, RNAs generated from both the AAV5 P7 and P19 promoters were efficiently polyadenylated at a site lying within the intronic region in the center of the genome. Because P7- and P19-generated transcripts are polyadenylated at this site and not spliced, Rep78 and Rep52 were the only Rep proteins detected during AAV5 infection.

The trypsin-sensitive RVER domain in the capsid proteins of minute virus of mice is required for efficient cell binding and viral infection but not for proteolytic processing in Vivo

Analysis of a series of mutations in the trypsin-sensitive RVER region of the amino terminal domain in the capsid proteins (VP1 and VP2) of the autonomous parvovirus, minute virus of mice (MVM), demonstrates that this sequence is not essential for proteolytic processing of VP2 into VP3 in vivo, but specific amino acids within this domain are important for viral infection. Analysis of the most deficient of these mutants, VP(delta 2842-2863), a 7-aa deletion of aa 159-165 in VP1 and 17-23 in VP2, has identified at least two steps in MVM infection in which this domain is important. VP(delta 2842-2863) was 3-fold defective in binding to murine A9(2L) cells and, when an equivalent amount of virus was bound to cells, additionally 10-fold deficient compared to wild-type in initiating a productive infection. However, in those cells effectively infected, VP(delta 2843-2863) replicated similar to wild-type. These results suggest that these seven amino acids constitute a region important for both binding and a subsequent step prior to the start of DNA replication such as viral uptake or transport to the nucleus.

Phenotypic Characterization of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis

Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited neurodegenerative disorder that results from mutations in the CLN3 gene. JNCL is characterized by accumulation of autofluorescent lysosomal storage bodies, vision loss, seizures, progressive cognitive and motor decline, and premature death. Studies were undertaken to characterize the neuronal ceroid lipofuscinosis phenotype in a Cln3 knockout mouse model. Progressive accumulation of autofluorescent storage material was observed in brain and retina of affected mice. The Cln3(-/-) mice exhibited progressively impaired inner retinal function, altered pupillary light reflexes, losses of inner retinal neurons, and reduced brain mass. Behavioral changes included reduced spontaneous activity levels and impaired learning and memory. In addition, Cln3(-/-) mice had significantly shortened life spans. These phenotypic features indicate that the mouse model will be useful for investigating the mechanisms underlying the disease pathology in JNCL and provide quantitative markers of disease pathology that can be used for evaluating the efficacies of therapeutic interventions.

Behavioral assessment in mouse models of neuronal ceroid lipofuscinosis using a light-cued T-maze.

Learning impairment is a common feature of the neuronal ceroid lipofuscinoses (NCL), a family of lysosomal storage disorders associated with progressive neurodegeneration. Murine models for the neuronal ceroid lipofuscinoses include the well-characterized motor neuron degeneration (mnd/mnd) model for one variant of late infantile NCL (CLN8), and the more recently generated models for the infantile (CLN1) and juvenile (CLN3) forms of NCL. To determine whether these mouse models exhibit behavioral deficits analogous to the learning impairment characteristic of the human disorders, the performance of these animals on an associative learning task was assessed. The abilities of affected and normal control mice to associate a light stimulus with a food reward were evaluated in 14-16-week-old animals using a T-maze. Normal mice were able to reach a criterion for having learned to make the association within a mean of 9.4 trials. The CLN8 and CLN3 mice, on the other hand, required means of 26.2 and 27.5 trials, respectively, to reach the same performance criterion (p<0.05), whereas none of the CLN1 mice were able to reach the criterion within a limit of 30 trials. The poor performance of the mutant mice did not appear to result from impaired retinal function; mice of all three strains exhibited retinal electrophysiological responses to dim light flashes and displayed robust pupillary light reflexes. Associative learning deficits appear to be an early disease phenotype in the NCL mouse models that will be useful for assessing the efficacy of therapeutic interventions such as gene or stem cell therapies.

Ocular phenotype in a mouse gene knockout model for infantile neuronal ceroid lipofuscinosis

Mutations in the human protein palmitoyl thioesterase‐1 (PPT‐1) gene result in an autosomal recessive neurodegenerative disorder designated neuronal ceroid lipofuscinosis (NCL), type CLN1, or infantile NCL. Among the symptoms of the CLN1 disease are accumulation of autofluorescent lysosomal storage bodies in neurons and other cell types, seizures, motor and cognitive decline, blindness, and premature death. Development of an effective therapy for this disorder will be greatly assisted by the availability of suitable animal models. A mouse PPT‐1 gene knockout model has recently been generated. Studies were performed to determine whether the mouse model exhibits ocular features of the human CLN1 disorder. A progressive accumulation of autofluorescent storage material in all layers of the retina was observed in the PPT‐1 knockout mice. Accompanying the storage body accumulation was a modest loss of cells with nuclei in the outer and inner nuclear layers. As indicated by electroretinogram (ERG) responses, retinal function was only mildly impaired at 4 months of age but was severely impaired by 8 months, despite only modest changes in retinal morphology. The pupillary light reflex (PLR), on the other hand, was exaggerated in the knockout mice. The apparent anomaly between the ERG and the PLR findings suggests that disease‐related PLR changes may be due to changes in extraocular signal processing. The pronounced ocular phenotype in the PPT‐1 knockout mice makes these animals a good model for testing therapeutic interventions for treatment of the human CLN1 disorder.

Immunological Barriers to Stem Cell Therapy in the Central Nervous System

The central nervous system is vulnerable to many neurodegenerative disorders such as Alzheimers disease that result in the extensive loss of neuronal cells. Stem cells have the ability to differentiate into many types of cells, which make them ideal for treating such disorders. Although stem cell therapy has shown some promising results in animal models for many brain disorders it has yet to translate into the clinic. A major hurdle to the translation of stem cell therapy into the clinic is the immune response faced by stem cell transplants. Here, we focus on immunological and related hurdles to stem cell therapies for central nervous system disorders.

Detection of Calcium Transients in Embryonic Stem Cells and Their Differentiated Progeny

A central issue in stem cell biology is the determination of function and activity of differentiated stem cells, features that define the true phenotype of mature cell types. Commonly, physiological mechanisms are used to determine the functionality of mature cell types, including those of the nervous system. Calcium imaging provides an indirect method of determining the physiological activities of a mature cell. Camgaroos are variants of yellow fluorescent protein that act as intracellular calcium sensors in transfected cells. We expressed one version of the camgaroos, Camgaroo-2, in mouse embryonic stem (ES) cells under the control of the CAG promoter system. Under the control of this promoter, Camgaroo-2 fluorescence was ubiquitously expressed in all cell types derived from the ES cells that were tested. In response to pharmacological stimulation, the fluorescence levels in transfected cells correlated with cellular depolarization and hyperpolarization. These changes were observed in both undifferentiated ES cells as well as ES cells that had been neurally induced, including putative neurons that were differentiated from transfected ES cells. The results presented here indicate that Camgaroo-2 may be used like traditional fluorescent proteins to track cells as well as to study the functionality of stem cells and their progeny.

Effective Treatment for the Canine RPE65 Null Mutation, a Hereditary Retinal Dystrophy Comparable to Human Leber’s Congenital Amaurosis

Mutations in the human retinal pigment epithelial (RPE)65 gene underlie some forms of early childhood blindness, including a form of Leber’s congenital amaurosis (LCA), early-onset severe retinal dystrophy, and juvenile retinitis pigmentosa (RP) (1, 2, 3, 4). LCA is an autosomal recessively inherited disease (5), although a few families with autosomal dominant inheritance have also been reported (6). In general, LCA is diagnosed when there is marked visual impairment from birth, whereas the disease is considered juvenile RP if vision is lost during the first 2 yr of life. Mutations in several genes other than RPE65 have also been identified in other ocular phenotypes designated as LCA (7).