Biomedical subjects

Howard J Federoff

Publications and source records attributed to Howard J Federoff.

At least 37 records · Page 2Linked to original sources

PubMed2005

HSV amplicon-mediated Abeta vaccination in Tg2576 mice: differential antigen-specific immune responses.

Given the participation of amyloid beta (Abeta) in Alzheimer's disease (AD) pathogenesis the derivation of experimental therapeutics to prevent Abeta fibrillogenesis and/or enhance removal of parenchymal amyloid deposits represent viable disease-modifying approaches. Active Abeta-based immunotherapies have shown promise in mouse AD models, but application in human trials was accompanied by moderate brain inflammation in a subset of patients. Immune-shaping vaccine platforms may mitigate adverse effects. Herein, we describe the use of herpes simplex virus (HSV)-derived amplicons to elicit distinctive immune responses against Abeta. Two vaccine vectors were constructed: one expressing Abeta1-42 alone (HSVAbeta), and a second expressing Abeta1-42 fused with the molecular adjuvant tetanus toxin Fragment C (HSVAbeta/TtxFC). Peripheral administration of these vaccines augmented humoral responses to Abeta and reduced CNS Abeta deposition in Tg2576 AD mice. Interestingly and unexpectedly, HSVAbeta vaccination was uniquely toxic and incited the expression of pro-inflammatory molecule transcripts within the hippocampi of Tg2576 mice, suggesting that this paradigm may serve as a relevant model to study Abeta vaccine-elicited CNS inflammatory syndromes.

Alzheimer Disease↗

PubMed2005

Human dendritic cells transduced with herpes simplex virus amplicons encoding human immunodeficiency virus type 1 (HIV-1) gp120 elicit adaptive immune responses from human cells engrafted into NOD/SCID mice and confer partial protection against HIV-1 challenge.

Small-animal models are needed to test human immunodeficiency virus (HIV) vaccine efficacy following viral challenge. To this end, we examined HIV-1-specific immune responses following immunization of nonobese diabetic-severe combined immunodeficient mice that were repopulated with human peripheral blood lymphocytes (hu-PBL-NOD/SCID mice). Autologous dendritic cells (DC) were transduced ex vivo with replication-defective, helper virus-free, herpes simplex virus type 1 (HSV-1) amplicons that expressed HIV-1 gp120 and were then injected into the hu-PBL-NOD/SCID mice. This resulted in primary HIV-1-specific humoral and cellular immune responses. Serum samples from vaccinated animals contained human immunoglobulin G that reacted with HIV-1 Env proteins by enzyme-linked immunosorbent assay and neutralized the infectivity of HIV-1 LAI and ADA strains. T cells isolated from the mice responded to viral antigens by producing gamma interferon when analyzed by enzyme-linked immunospot assay. Importantly, exposure of the vaccinated animals to infectious HIV-1 demonstrated partial protection against infectious HIV-1 challenge. This was reflected by a reduction in HIV-1(ADA) and by protection of the engrafted human CD4(+) T lymphocytes against HIV-1(LAI)-induced cytotoxicity. These data demonstrate that transduction of DC by HSV amplicon vectors expressing HIV-1 gp120 induce virus-specific immune responses in hu-PBL-NOD/SCID mice. This mouse model may be a useful tool to evaluate human immune responses and protection against viral infection following vaccination.

Animals↗

PubMed2005

Altered gene expression profiles reveal similarities and differences between Parkinson disease and model systems.

Parkinson disease (PD) targets dopaminergic neurons in the substantia nigra, resulting in motor disturbances such as resting tremor, bradykinesia, and rigidity. Pathogenic processes likely occur over several decades, in that an overwhelming percentage of neurons are already dead at the time of clinical diagnosis. For this reason, the usage of animal model systems to discover the early steps in the pathologic cascade is required. These include exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which selectively kills dopamine neurons in the substantia nigra, and genetic models incorporating mutations in the alpha-synuclein gene that cause disease in human patients. Through the evaluation of these models at multiple time points, it is possible to discover novel gene expression changes that may underlie disease pathogenesis. Specifically, the authors hypothesize that animal models of PD and human PD brains share a gene expression profile that signifies certain aspects of pathogenesis and/or recovery-resistance. To test this and similar hypotheses, the authors and others have utilized new microarray technology that enables the sampling of thousands of genes' expression level in one assay. Because the technology is fairly new and results can vary depending on methods used, results must be evaluated with care. Multiple array and data-mining options can be used to make the most accurate inferences as to differentially expressed genes in each set of samples. The authors developed a fusion classifier approach whereby individual data-mining algorithms generate lists of significant genes. The lists are subsequently queried, and only genes unanimously called significant are retained for further validation. Although the authors' approach identified hundreds of differentially expressed genes in each of three PD systems, only a few were common between the human and animal substantia nigra. These were related to dopamine phenotype, synaptic function, and the mitochondrial metabolism, implicating the presynaptic terminal as a primary site of injury. The time course of the authors' experiments indicates that if the synaptic changes could be prevented, this may alleviate some cell death, in that these changes precede neuronal loss.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2004

Apoptosis-inducing factor substitutes for caspase executioners in NMDA-triggered excitotoxic neuronal death.

The profound neuroprotection observed in poly(ADP-ribose) polymerase-1 (PARP-1) null mice to ischemic and excitotoxic injury positions PARP-1 as a major mediator of neuronal cell death. We report here that apoptosis-inducing factor (AIF) mediates PARP-1-dependent glutamate excitotoxicity in a caspase-independent manner after translocation from the mitochondria to the nucleus. In primary murine cortical cultures, neurotoxic NMDA exposure triggers AIF translocation, mitochondrial membrane depolarization, and phosphatidyl serine exposure on the cell surface, which precedes cytochrome c release and caspase activation. NMDA neurotoxicity is not affected by broad-spectrum caspase inhibitors, but it is prevented by Bcl-2 overexpression and a neutralizing antibody to AIF. These results link PARP-1 activation with AIF translocation in NMDA-triggered excitotoxic neuronal death and provide a paradigm in which AIF can substitute for caspase executioners.

Active Transport, Cell Nucleus↗

PubMed2004

Dysregulation of gene expression in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse substantia nigra.

Parkinson's disease pathogenesis proceeds through several phases, culminating in the loss of dopaminergic neurons of the substantia nigra (SN). Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of oxidative SN injury is frequently used to study degeneration of dopaminergic neurons in mice and non-human primates, an understanding of the temporal sequence of molecular events from inhibition of mitochondrial complex 1 to neuronal cell death is limited. Here, microarray analysis and integrative data mining were used to uncover pathways implicated in the progression of changes in dopaminergic neurons after MPTP administration. This approach enabled the identification of small, yet consistently significant, changes in gene expression within the SN of MPTP-treated animals. Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity, and cell cycle and apoptosis. The discovery and validation of these alterations provide molecular evidence for an evolving cascade of injury, dysfunction, and cell death.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2004

Viral delivery of NR2D subunits reduces Mg2+ block of NMDA receptor and restores NT-3-induced potentiation of AMPA-kainate responses in maturing rat motoneurons.

N-methyl-D-aspartate (NMDA) responsiveness of motoneurons declines during the initial 2 postnatal weeks due to increasing Mg2+ block of NMDA receptors. Using gene chip analyses, RT-PCR, and immunochemistry, we have shown that the NR2D subunit of the NMDA receptor (NMDAR), known to confer resistance to Mg2+ block, also declines in motoneurons during this period. We injected a viral construct (HSVnr2d) into the lumbar spinal cord on postnatal day 2 in an attempt to restore NMDAR function in motoneurons during the second postnatal week. Following HSVnr2d injection, we detected elevated levels of NR2D mRNA in spinal cord samples and NR2D protein specifically in motoneurons. These molecular changes were associated with marked functional alterations whereby NMDAR-mediated responses in motoneurons associated with both dorsal root (DR) and ventrolateral funiculus (VLF) inputs returned to values observed at E18 due to decreased Mg2+ blockade. Viruses carrying the beta-galactosidase gene did not induce these effects. NT-3 is known to potentiate AMPA-kainate responses in motoneurons if the response has an NMDAR-mediated component and thus is normally ineffective during the second postnatal week. Restoration of NMDAR-mediated responsiveness in the second postnatal week was accompanied by a return of the ability of neurotrophin-3 (NT-3) to potentiate the AMPA-kainate responses produced by both DR and VLF synaptic inputs. We conclude that delivery of the gene for a specific NMDA subunit can restore properties characteristic of younger animals to spinal cord motoneurons. This approach might be useful for enhancing the function of fibers surviving in the damaged spinal cord.

Animals↗

PubMed2004

Herpes simplex virus amplicon delivery of a hypoxia-inducible angiogenic inhibitor blocks capillary formation in hepatocellular carcinoma.

Tumor hypoxia induces vascular endothelial growth factor (VEGF) expression, which stimulates tumor angiogenesis. The VEGF pathway is inhibited by soluble VEGF receptors (soluble fetal liver kinase-1 [sFlk-1]) that bind VEGF and block its interaction with endothelial cells. Herpes simplex virus (HSV)-derived amplicons are replication-incompetent viruses used for gene delivery. We attempt to attenuate angiogenesis and inhibit hepatoma growth through amplicon-mediated expression of sFlk-1 under hypoxic control. A multimerized hypoxia-responsive enhancer (10xHRE) was cloned upstream of the sFlk-1 gene (10xHRE/sFlk-1). An amplicon expressing 10xHRE/sFlk-1 was genetically engineered (HSV10xHRE/sFlk-1). SK-HEP-1 human hepatoma cells were transduced with HSV10xHRE/sFlk-1 and incubated in normoxia (21% O2) or hypoxia (1% O2). Human umbilical vein endothelial cell assay evaluated capillary inhibition. Western blot assessed sFlk-1 expression. SK-HEP-1 flank tumors (n = 24) in athymic mice were treated with HSV10xHRE/sFlk-1. Media from hypoxic SK-HEP-1 transduced with HSV10xHRE/sFlk-1 yielded an 80% reduction in capillary formation (P < 0.005), whereas normoxic SK-HEP-1 yielded a 25% reduction (P < 0.05). Western blot of SK-HEP-1 transduced with HSV10xHRE/sFlk-1 demonstrated greater sFlk-1 expression in hypoxia vs. normoxia. SK-HEP-1 tumors treated with HSV10xHRE/sFlk-1 yielded a 72% reduction in volume vs. the control group (P < 0.000001). HSV amplicon-mediated delivery of a hypoxia-inducible soluble VEGF receptor substantially reduces new vessel formation and tumor growth in hepatoma.

Angiogenesis Inhibitors↗

PubMed2004

The role of the THY1 gene in human ovarian cancer suppression based on transfection studies.

In our recent studies, the expression of the THY1 gene encoding a 25-28 kDa glycoprotein located at 11q23-q24, was found to be associated with complete tumor suppression of the ovarian cancer cell line SKOV-3 after the transfer of chromosome 11. These studies raised the possibility that THY1 maybe a candidate tumor suppressor gene for ovarian cancer. To investigate this, the complete cDNA sequence for THY1 was cloned and transfected into SKOV-3 ovarian cancer cells. The expression of THY1 in the transfectants was confirmed by Northern blot analysis, immunocytochemistry, and flow cytometry. Both SKOV-3-THY1 and SKOV-3-null cells were inoculated subcutaneously into severe combined immunodeficiency (SCID) mice to determine in vivo tumorigenicity. THY1 transfectants formed tumors, but overall tumor growth rate and tumor size was significantly reduced compared with their null counterparts. To further correlate THY1 expression with tumorigenicity, the THY1 antisense was transfected into the nontumorigenic clone, 11(C)9-8, which resulted in restoration of tumorigenicity. These data indicate that THY1 expression alone cannot suppress tumorigenicity; however, abrogation of THY1 expression from nontumorigenic cells can restore tumorigenesis. Taken together, the data suggest that THY1 is necessary but not sufficient to suppress ovarian tumorigenicity. Therefore, THY1 can be designated as a putative tumor suppressor gene for human ovarian cancer.

Animals↗

PubMed2004

Utilizing tumor hypoxia to enhance oncolytic viral therapy in colorectal metastases.

OBJECTIVE: To determine the effects of hypoxia-induced ribonucleotide reductase (RR) production on herpes oncolytic viral therapy. SUMMARY BACKGROUND DATA: Hypoxia is a common tumor condition correlated with therapeutic resistance and metastases. Attenuated viruses offer a unique cancer treatment by specifically infecting and lysing tumor cells. G207 is an oncolytic herpes virus deficient in RR, a rate-limiting enzyme for viral replication. METHODS: A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested by luciferase assay. 10xHRE was cloned upstream of UL39, the gene encoding the large subunit of RR (10xHRE-UL39). CT26 murine colorectal cancer cells were transfected with 10xHRE-UL39, incubated in hypoxia (1% O2) or normoxia (21% O2), and infected with G207 for cytotoxicity assays. CT26 liver metastases, with or without 10xHRE-UL39, were created in syngeneic Balb/C mice (n = 40). Livers were treated with G207 or saline. Tumors were assessed and stained immunohistochemically for G207. RESULTS: 10xHRE increased luciferase expression 33-fold in hypoxia versus controls (P < 0.001). In normoxia, 10xHRE-UL39 transfection did not improve G207 cytotoxicity. In hypoxia, G207 cytotoxicity increased 87% with 10xHRE-UL39 transfection versus nontransfected cells (P < 0.001). CT26 were resistant to G207 alone. Combining 10xHRE-UL39 with G207 resulted in a 66% decrease in tumor weights (P < 0.0001) and a 65% reduction in tumor nodules (P < 0.0001) versus G207 monotherapy. 10xHRE-UL39-transfected tumors demonstrated greater viral staining. CONCLUSIONS: Hypoxia-driven RR production significantly enhances viral cytotoxicity in vitro and reduces tumor burden in vivo. G207 combined with RR under hypoxic control is a promising treatment for colorectal cancer, which would otherwise be resistant to oncolytic herpes virus alone.

Animals↗

PubMed2004

Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression.

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.

Animals↗

PubMed2004

Safety of viral vectors for neurological gene therapies.

Viral-based gene therapy for neurological diseases provides a daunting challenge. Many neurological diseases, especially neurodegenerative conditions, have unknown etiologies and current viral platforms are largely inadequate for human therapy. The challenges appear surmountable through systematic definition and solution. This review highlights three problem areas for gene therapy; genomic integration, immunogenic responses and regulated gene expression, as well as current progress in those areas. Successful neurological gene therapy will require efficacy along with clear biosafety. Different vector platforms manifest characteristic properties that affect their suitability for human gene therapy and among these are their propensity to integrate, elicit immune responses and enable regulated gene expression.

Gene Expression Regulation↗

PubMed2003

Systemic FIV vector administration: transduction of CNS immune cells and Purkinje neurons.

The systemic effects of gene therapy have been previously described in a variety of peripheral organs following intravenous administration or intraperitoneal inoculation of viral vectors, as well as in the brain following intracranial administration. However, limited information is available on the ability of viral vectors to cross the blood-brain barrier and infect cells located within the central nervous system (CNS). We employed a VSV-G pseudotyped FIV(lacZ) vector capable of transducing dividing, growth-arrested, as well as post-mitotic cells with the reporter gene lacZ. Adult mice were injected intraperitoneally with FIV(lacZ), and the expression of beta-galactosidase was studied 5 weeks following treatment in the brain, liver, spleen and kidney by X-gal histochemistry and immunocytochemistry. Interestingly, relatively low doses of FIV(lacZ) administered intraperitoneally lead to beta-galactosidase detection in the brain and cerebellum. The identity of these cells was confirmed by double immunofluorescence, and included CD31-, CD3- and CD11b-positive cells. Fluorescent microspheres co-injected with FIV(lacZ) virus were identified within mononuclear cells in the brain parenchyma, suggesting infiltration of peripheral immune cells in the CNS. Cerebellar Purkinje neurons were also transduced in all adult-injected mice. Our observations indicate that relatively low doses of FIV(lacZ) administered intraperitoneally resulted in the transduction of immune cells in the brain, as well as a specific subset of cerebellar neurons.

Animals↗

PubMed2003

p75 neurotrophin receptor protects primary cultures of human neurons against extracellular amyloid beta peptide cytotoxicity.

The cytotoxicity of extracellular amyloid beta peptide (Abeta) has been clearly demonstrated in many cell types. In contrast, primary human neurons in culture are resistant to extracellular Abeta-mediated toxicity. Here, we investigate the involvement of p75 neurotrophin receptor (p75NTR) in Abeta-treated human neurons. We find that Abeta1-40 and Abeta1-42, but not the reverse control peptide, Abeta40-1, rapidly increase the levels of p75NTR in a specific and dose-dependent manner. In contrast to observations in cell lines, enhanced expression of p75NTR in human neurons via a herpes simplex virus amplicon vector does not increase the susceptibility of neurons to Abeta. Unexpectedly, inhibition of p75NTR expression with an antisense expression construct or incubation of the cells with an antibody to the extracellular domain of p75NTR sensitizes human neurons to extracellular nonfibrillar or fibrillar Abeta1-42 cytotoxicity. Unlike intracellular Abeta, extracellular Abeta toxicity is independent of p53 and Bax activity. However, Abeta toxicity is inhibited by caspase inhibitors and the glycogen synthase kinase 3beta inhibitor lithium. Neuroprotection against Abeta is phosphatidylinositide 3-kinase dependent but Akt independent. These results are consistent with a neuroprotective role for p75NTR against extracellular Abeta toxicity in human neurons.

Amyloid beta-Peptides↗

PubMed2003

Cellular immune responses to helper-free HSV-1 amplicon particles encoding HIV-1 gp120 are enhanced by DNA priming.

A single inoculation of herpes simplex virus type-1 (HSV-1) amplicon vectors encoding human immunodeficiency virus type-1 gp120 (HSV:gp120) results in robust, specific immune responses to gp120. To explore further the utility of this novel vaccine delivery system, we examined the kinetics of the cellular immune response by tetramer staining, following a single intramuscular administration of HSV:gp120 particles, and found that it peaks at 9-28 days post-immunization, before declining to a stable memory response. We also examined the utility of prime-boost regimens using packaged amplicon particles and naked amplicon plasmid DNA (DNA:gp120). These experiments showed that two sequential immunizations with HSV:gp120 resulted in a 5-10-fold increase in gp120-specific cellular immune responses and that plasmid DNA priming, followed by amplicon particle boosting, imparted the strongest acute and memory T cell responses, as determined by tetramer analysis. Collectively, these results demonstrate the utility of HSV amplicon vectors in prime-boost regimens for HIV vaccine development.

Animals↗

PubMed2003

Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing.

Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli beta-galactosidase in mouse or human target cells, and an Elispot to detect release of beta-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-gamma secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.

Animals↗

PubMed2003

Neurotrophin secretory pathways and synaptic plasticity.

Neurotrophins and synaptic activity work in conjunction during the process of synaptic plasticity. Both are required to stabilize new synaptic structures and the loss of either can lead to cognitive impairments, as new information cannot be stored efficiently or later recalled. Neurotrophins are becoming recognized as mediators of activity-dependent plasticity. The synthesis and release of neurotrophins follow neurosecretory pathways. They are synthesized as immature, pro-neurotrophin molecules, processed, and then mature forms are secreted. These then activate distinct signal tranduction pathways that modify the synapse. High frequency stimulation is known to induce long-term potentiation (LTP) and synaptic enhancement, however, the stability of these changes requires a bi-directional communication between the pre- and post-synaptic terminals and neurotrophins can function in such a capacity. The NGFXAT somatic mosaic murine model demonstrated how neurotrophin and active learning induces a robust synaptic reorganization. Synaptic dysfunction decreases efficient neurotransmission or neurotrophin production and then precedes overt neurodegeneration. The study of synaptic dysfunction and neurotrophin actions with respect to activity will lead to future therapeutic interventions of age-related dementias.

Animals↗

PubMed2003

Transcriptional and posttranslational regulation of Cre recombinase by RU486 as the basis for an enhanced inducible expression system.

Genetic studies often require the employment of an inducible expression system, whereby the expression of a particular gene can be regulated by the exogenous administration of an inert ligand. Cre/loxP-based systems have been previously described as the basis for inducible expression systems by exerting site-specific DNA recombination. In our effort to enhance the properties of the RU486-responsive CrePr1 construct, we have developed the dual GLVP/CrePr system, in which RU486 confers activity control at both the transcriptional and the posttranslational level of CrePr1. This was achieved by placing CrePr1 transcriptional regulation under the control of the RU486-sensitive chimeric regulator GLVP. Stable cell lines harboring the dual GLVP/CrePr as well as the single CrePr1 system were developed. Our results indicate that the dually regulated system is highly inducible by RU486 while maintaining minimal basal activity ("leakage"), characteristics that can be employed in the development of transgenic mice in which genetic pathways can be turned on or turned off after exogenous administration of RU486 at physiologically inert doses.

Animals↗

PubMed2003

Helper-free HSV-1 amplicons elicit a markedly less robust innate immune response in the CNS.

The development and implementation of direct gene transfer technologies for the study and treatment of chronic CNS disorders inherently requires consideration of vector safety. Virus-based vectors represent the most efficient modalities but harbor the potential to induce vigorous innate and adaptive immune responses when administered in vivo. These responses can arise because of virus particle components, resultant viral gene expression, and/or transgene expression. In the current study, we describe the innate responses elicited upon stereotactic delivery of herpes simplex virus type 1-based amplicon vectors. C57BL/6 mice were injected with sterile saline, beta-galactosidase-expressing amplicon (HSVlac) packaged by a conventional helper virus-based technique, or helper virus-free HSVlac. After killing the mice at either 1 or 5 days after transduction, we analyzed them by immunocytochemistry and quantitative RT-PCR for various chemokine, cytokine, and adhesion molecule gene transcripts. All injections induced inflammation, with blood/brain barrier opening on day 1 that was enhanced with both amplicon preparations as compared with saline controls. By day 5, mRNA levels for the pro-inflammatory cytokines (IL-1beta, TNF-alpha, IFN-gamma), chemokines (MCP-1, IP-10), and an adhesion molecule (ICAM-1) had returned to baseline in saline-injected mice and to near-baseline levels in helper virus-free amplicon groups. In contrast, mice injected with helper virus-packaged amplicon stocks elicited elevated inflammatory molecule expression and immune cell infiltration even at day 5. In aggregate, we demonstrate that helper virus-free amplicon preparations exhibit a safer innate immune response profile, presumably as a result of the absence of helper virus gene expression, and provide support for future amplicon-based CNS gene transfer strategies.

3T3 Cells↗