The central nervous system is vulnerable to many neurodegenerative disorders such as Alzheimer's 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.

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.

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.

The cat has served as an important nonrodent research model for neurophysiology and retinal degenerative disease processes, yet very little is known about feline neural precursor cells. To culture these cells and evaluate marker expression, brains were dissected from 45-day-old fetuses, enzymatically dissociated, and grown in the presence of EGF, bFGF and PDGF. Expanded cells widely expressed nestin, Sox2, Ki-67, fusin (CXCR4) and vimentin, while subpopulations expressed A2B5, GFAP, or beta-III tubulin. Precursors prelabeled with BrdU and/or transduced with a recombinant lentivirus that expresses GFP were transplanted subretinally in five dystrophic Abyssinian cats. Two to 4 weeks following surgery, histology showed survival of grafted cells in three of the animals. Labeled cells were found in the neuroretina and RPE layer, as well as in the vitreous and the vicinity of Bruch's membrane. There was no evidence of an immunologic response in any of the eyes. Neural precursor cells can therefore be cultured from the developing cat brain and survive as allografts for up to 4 weeks without immune suppression. The feasibility of deriving and transplanting feline neural precursor cells, combined with the availability of the dystrophic Abyssinian cat, provide a new feline model system for the study of retinal repair.

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) (1–4). 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).

Spinal muscular atrophy (SMA) is caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene called SMN2 is present in all SMA patients; however SMN2 produces low levels of functional protein due to alternative splicing. Recently a therapeutic approach has been developed referred to as trans-splicing. Conceptually, this strategy relies upon pre-messenger RNA (pre-mRNA) splicing occurring between two separate molecules: (i) the endogenous target RNA and (ii) the therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. SMN trans-splicing RNAs were initially examined and expressed from a plasmid-backbone and shown to re-direct splicing from a SMN2 mini-gene as well as from endogenous transcripts. Subsequently, recombinant adeno-associated viral vectors were developed that expressed and delivered trans-splicing RNAs to SMA patient fibroblasts. In the severe SMA patient fibroblasts, SMN2 splicing was redirected via trans-splicing to produce increased levels of full-length SMN mRNA and total SMN protein levels. Finally, small nuclear ribonucleoprotein (snRNP) assembly, a critical function of SMN, was restored to SMN-deficient SMA fibroblasts following treatment with the trans-splicing vector. Together these results demonstrate that the alternatively spliced SMN2 exon 7 is a tractable target for replacement by trans-splicing.

Recombinant AAV vectors are produced by transient transfection of mammalian cells. The virus is usually purified from a combination of lysed cells and spent culture medium by HPLC. We have developed a quantitative, real-time PCR assay for quantifying encapsidated single-stranded viral DNA (i.e. DNA-containing virions) in cell lysates and the spent culture medium. This requires extensive DNaseI digestion to reduce the amount of AAV replicative DNA, as well as plasmid and cellular DNA, to negligible amounts. To demonstrate the utility of this assay, we produced recombinant AAV in HeLa cells and five different types of 293 cells. We used primers to the EGFP transgene to detect the production of a recombinant AAV. We assayed the cell lysates and media by both our quantitative PCR assay and a functional transduction assay. The quantitative PCR assay data correlated well with the transduction assay data. Because this assay only requires standard PCR primers and SYBR Green I dye to detect the amplification of the PCR template, it will readily adapt to any target DNA sequence within the recombinant AAV genome. The recombinant AAV vector does not need to express a reporter gene, such as EGFP or β-galactosidase in order to assay the amount of virus produced.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present, called SMN2. SMN2 is retained in all SMA patients and encodes an identical protein compared to SMN1. However, a single silent nucleotide difference in SMN2 exon 7 results in the production of a spliced isoform (called SMNDelta7) that encodes a nonfunctional protein. The presence of SMN2 represents a unique therapeutic target since SMN2 has the capacity to encode a fully functional protein. Here we describe an in vivo delivery system for short bifunctional RNAs that modulate SMN2 splicing. Bifunctional RNAs derive their name from the presence of two domains: an antisense RNA sequence specific to a target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. Plasmid-based and recombinant adeno-associated virus vectors were developed that expressed bifunctional RNAs that stimulated SMN2 exon 7 inclusion and full-length SMN protein in patient fibroblasts. These experiments provide a mechanism to modulate splicing from a variety of genetic contexts and demonstrate directly a novel therapeutic approach for SMA.

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. © 2006 Wiley‐Liss, Inc.

We have developed a very simple protocol to transfect mammalian cells with linear polyethylenimine (PEI). Linear PEI has been used to transfect nucleic acids into a wide variety of mammalian cells both in cell culture and in vivo. Almost all of these reports have used commercially available preparations of PEI. Our linear PEI protocol is as efficient as commercial reagents in the transfection of HEK293 and HeLa cells, but at only a fraction of the cost. Our protocol should be useful for many different applications. It should be particularly useful for large-scale production of recombinant proteins and viruses requiring transfection of mammalian cells in large batches. We have used this protocol to produce recombinant AAV, which requires transfection of cells with three plasmids simultaneously. One plasmid contains the recombinant AAV genome. The second plasmid expresses the AAV rep and cap genes. The third plasmid expresses several adenovirus genes that are required for AAV replication. We have assessed production of a recombinant AAV that expresses GFP both by quantitative PCR and by transduction of HeLa cells.

We have previously reported on successful gene transfer using recombinant adeno-associated virus-mediated (rAAV) subretinal gene delivery for the dog model of human Leber's Congenital Amaurosis. Congenitally blind dogs were treated at 4-30 months of age with similar doses of the rAAV. RPE65 construct into the right eye.

Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovirus-infected cells by cotransfecting a plasmid containing the recombinant AAV2 genome, which is generally comprised of the viral terminal repeats flanking a transgene, together with a second plasmid expressing the AAV2 rep and cap genes. However, recombinant viruses generally replicate inefficiently, often producing 100-fold fewer virus particles per cell than can be obtained after transfection with a plasmid containing a wild-type AAV2 genome. We demonstrate that this defect is due, at least in part, to the presence of a positive-acting cis element between nucleotides 194 and 1882 of AAV2. Recombinant AAV2 genomes lacking this region accumulated 14-fold less double-stranded, monomer-length replicative-form DNA than did wild-type AAV2. In addition, we demonstrate that a minimum genome size of 3.5 kb is required for efficient production of single-stranded viral DNA. Relatively small recombinant genomes (2,992 and 3,445 bp) accumulated three- to eightfold less single-stranded DNA per monomer-length replicative-form DNA molecule than wild-type AAV2. In contrast, recombinant AAV2 with larger genomes (3,555 to 4,712 bp) accumulated similar amounts of single-stranded DNA per monomer-length replicative-form DNA compared to wild-type AAV2. Analysis of two recombinant AAV2 genomes less than 3.5 kb in size indicated that they were deficient in the production of the extended form of monomer-length replicative-form DNA, which is thought to be the immediate precursor to single-stranded AAV2 DNA.

The adenovirus 12S and 13S E1A proteins have been shown to relieve repression mediated by the cellular transcription factor YY1. The 13S E1A protein not only relieves repression but also activates transcription through YY1 binding sites. In this study, using a variety of in vivo and in vitro assays, we demonstrate that both E1A proteins can bind to YY1, although the 13S E1A protein binds more efficiently than the 12S E1A protein. Two domains on the E1A proteins interact with YY1: an amino-terminal sequence (residues 15 to 35) that is present in both E1A proteins and a domain that includes at least a portion of conserved region 3 (residues 140 to 188) that is present in the 13S but not the 12S E1A protein. Two domains on YY1 interact with E1A proteins: one is contained within residues 54 to 260, and the other is contained within the carboxy-terminal domain of YY1 (residues 332 to 414). Cotransfection of a plasmid expressing carboxy-terminal amino acids 332 to 414 of YY1 fused to the GAL4 DNA-binding domain can inhibit expression from a reporter construct with GAL4 DNA binding sites in its promoter, and inclusion of a third plasmid expressing E1A proteins can relieve the repression. Thus, we find a correlation between the ability of E1A to interact with the carboxy-terminal domain of YY1 and its ability to relieve repression caused by the carboxy-terminal domain of YY1. We propose that E1A proteins normally relieve YY1-mediated transcriptional repression by binding directly to the cellular transcription factor.

We have characterized the temporal appearance and accumulation of minute virus of mice (MVM) replicative forms (RF) in highly synchronized single rounds of infection using a combination of restriction endonuclease analysis and two-dimensional agarose gel electrophoresis. Between 4 and 12 h after release of infected cells into the S-phase, both monomer (mRF) and dimer RF (dRF) increased exponentially at similar rates such that the ratio of mRF relative to dRF remained unchanged. These DNA forms accumulated at a faster rate than MVM RNAs, suggesting that the number of DNA templates available for replication is limiting, not the expression of MVM gene products, and that the majority of DNA templates are likely to be destined for DNA amplification rather than transcription and further gene expression. During this exponential DNA amplification phase, approximately 65 % of mRF were in a fully extended form, whereas most of the remaining mRF were covalently closed in the left end and extended in the right end. Although MVM replication presumably generates right-hand turn-around mRF, only a low level of this form persists (5 to 10 % of total mRF) at all times examined, suggesting that this form must be quickly converted to the extended form. Greater than 90 % of dRF, which have right-hand palindromes on both ends of the molecule, were extended on both ends. A significant proportion of dRF and higher concatemers are nicked in the left-hand palindrome, suggesting that resolution of dRF into two mRFs may occur via single-stranded nicks rather than a double-stranded cut. An additional replicative form, previously termed band X, has been identified as an RNA-DNA duplex. This band is formed predominantly intracellularly, before cell lysis but its biological significance remains unclear. Our results provide direct experimental support for many of the predictions of the current models of parvovirus replication and suggest that the kinetic hairpin transfer model should be adjusted to include a strand-transfer or similar mechanism for the resolution of dRF to account adequately for the production of left-end turn-around forms.