Mary Garrity-Moses

A novel peptide defined through phage display for therapeutic protein and vector neuronal targeting

A novel peptide with the binding characteristics of tetanus toxin was identified with phage display, for application in therapeutic protein and vector motor and sensory neuron targeting. A 12mer phage library was biopanned on trisialoganglioside (G(T1b)) and eluted with the tetanus toxin C fragment (rTTC). Phage ELISAs revealed increases in G(T1b) binding for the Tet1 and Tet2 phage clones when compared to peptideless phage (PLP). rTTC displaced both Tet1 and Tet2 phage clones from G(T1b), and both clones reduced rTTC-G(T1b) binding. Comparison of Tet1, Tet2, PLP, and the random phage library binding to PC12 and HEK293 cells revealed enhanced cellular binding by Tet1 and Tet2 phage. Tet1 phage binding was selective for neurons. Immunofluorescence also confirmed selective PC12 binding of Tet1 and Tet2 phage. Fluorescein-conjugated synthetic Tet1, but not Tet2, peptide showed strong binding to cultured PC12, primary motor neurons, and dorsal root ganglion (DRG) cells. Synthetic Tet1 bound DRG and motor neurons but not muscle in tissue sections. The enhanced neuronal binding affinity and specificity of Tet1, a novel 12 amino acid peptide, suggests potential utility for targeting neurotherapeutic proteins and viral vectors in the treatment of motor neuron disease, neuropathy, and pain.

Targeted spinal cord therapeutics delivery: stabilized platform and microelectrode recording guidance validation.

Background/Aims: No validated delivery technique exists for accurate, reproducible delivery of biological therapies to discrete spinal cord targets. To address this unmet need, we have constructed a stabilized platform capable of supporting physiologic mapping, through microelectrode recording, and cellular or viral payload delivery to the ventral horn. Methods: A porcine animal model (n = 7) has been chosen based upon the inherent morphologic similarities between the human and porcine spine. Animals underwent physiologic mapping and subsequent microinjection of a green-fluorescent-protein-labeled cell suspension. Sacrifice (t = 3 h) was performed immediately following behavioral assessment. Results: Histologic analysis has supported our ability to achieve localization to the ipsilateral ventral horn in the spinal cord. Complications included death due to malignant hyperthermia (n = 1), hindlimb dysfunction attributable to epidural hematoma (n = 1), and hindlimb dysfunction attributable to cord penetration (n = 2). Conclusions: These results indicate an ability to achieve accurate targeting, but the elevated incidence of neurologic morbidity will require further studies with longer follow-ups that incorporate procedural and equipment modifications that will allow for a reduced number of cord penetrations and will account for observed cardiorespiratory-associated cord movement. These initial results reinforce the challenges of translating biological restorative therapies from small to large animal models and ultimately to humans.

Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination

Clostridial neurotoxins have assumed increasing importance in clinical application. The toxins light chain component (LC) inhibits synaptic transmission by digesting vesicle-docking proteins without directly altering neuronal health. To study the properties of LC gene expression in the nervous system, an adenoviral vector containing the LC of tetanus toxin (AdLC) was constructed. LC expressed in differentiated neuronal PC12 cells was shown to induce time- and concentration-dependent digestion of mouse brain synaptobrevin in vitro as compared to control transgene products. LC gene expression in the rat lumbar spinal cord disrupted hindlimb sensorimotor function in comparison to control vectors as measured by the Basso–Beattie–Bresnahan (BBB) scale (P<0.001) and rotarod assay (P<0.003). Evoked electromyography (EMG) showed increased stimulus threshold and decreased response current amplitude in LC gene-transferred rats. At the peak of functional impairment, neither neuronal TUNEL staining nor reduced motor neuron density could be detected. Spontaneous functional recovery was observed to parallel the cessation of LC gene expression. These results suggest that light chain gene delivery within the nervous system may provide a nondestructive means for focused neural inhibition to treat a variety of disorders related to excessive synaptic activity, and prove useful for the study of neural circuitry.

Neuroprotective adeno-associated virus Bcl-xL gene transfer in models of motor neuron disease

Recent work implicates excitotoxicity‐induced apoptosis as the mechanism triggering motor neuron death in amyotrophic lateral sclerosis (ALS). Our laboratory has previously utilized glutamate excitotoxicity in vitro to study this process. The present experiment tests whether overexpression of the gene for Bcl‐XL can inhibit excitotoxicity in this model system. To track Bcl‐XL expression, the gene for green fluorescent protein (GFP) was inserted in‐frame, upstream of the Bcl‐XL gene. The GFP‐Bcl‐XL gene was then cloned into an adeno‐associated viral (AAV2) vector. GFP expression in both SH‐SY5Y and embryonic day 15 (E15) motor neurons (MNs) peaked 48 hours after infection. Bcl‐XL expression in SH‐SY5Y cells significantly reduced terminal deoxy‐UTP nick‐end labeling (TUNEL)–positive cells and maintained cell density after glutamate exposure. Similarly, Bcl‐XL expression inhibited the development of TUNEL staining in E15 MNs and supported cell density after glutamate exposure. These findings suggest that AAV‐mediated expression of genes for antiapoptotic proteins may provide a means for ALS gene therapy. Muscle Nerve, 2005

X-linked inhibitor of apoptosis protein gene-based neuroprotection for the peripheral nervous system

OBJECTIVE The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of programmed cell death. In the current experiment, we examine the potential of adenoviral XIAP gene delivery to protect neurons of the peripheral nervous system using in vitro models of amyotrophic lateral sclerosis (ALS) and diabetic neuropathy. METHODS XIAP complementary deoxyribonucleic acid was fused in frame with the green fluorescent protein sequence and cloned into a first generation adenoviral vector. The impact of XIAP gene expression on glutamate-induced apoptosis was measured in the neuronal SH-SY5Y cell line with immunohistochemistry for active caspase-3 and with cell density assays. Next, the effect of XIAP expressing neurons on the survival of uninfected neighboring neurons was measured. Finally, the impact of XIAP gene expression on glutamate-induced apoptosis was assessed in embryonic motor neuron and dorsal root ganglion cultures. RESULTS XIAP gene expression reduced the percentage of active caspase-3 positive SH-SY5Y neurons and preserved cell density after glutamate exposure. In heterogeneously infected cultures, cells infected with XIAP were protected, but uninfected neighboring cells were not. In primary E15 models, inhibition of proapoptotic effects was demonstrated after glutamate insult in motor neurons and glucose insult in dorsal root ganglion cells. CONCLUSION XIAP gene delivery through the neurosurgical delivery of viral vectors may provide a means for neuroprotection in ALS and diabetic neuropathy.

Adeno-associated viral glutamate decarboxylase expression in the lateral nucleus of the rat hypothalamus reduces feeding behavior.

In vivo gene transfer of glutamate decarboxylase (GAD) has been explored as a means of inducing or increasing the production of the inhibitory amino-acid neurotransmitter, GABA. This strategy has been applied to neuroprotection, seizure prevention, and neuromodulation. In the present experiment, AAV2 was used to transfer the genes for green fluorescence protein (GFP) and GAD65 into the lateral nucleus of the rat hypothalamus. Microinjection of 500 nl of AAV2 resulted in transduction of a 0.25±0.04 mm3 with targeting errors of X=0.48 mm, Y=0.18 mm, Z=0.37 mm using standard stereotactic technique. Pre- and postinjection food and water consumption, urine and feces production, and weight were recorded. In comparison with rAAVCAGGFP- and PBS-injected animals, rats treated with rAAVCAGGAD65 demonstrated reduced weight gain (P<0.014) and transiently reduced daily food consumption (P<0.007) during the postoperative period. No changes in water consumption or waste production were recorded. Effective GAD65 gene transfer was confirmed with in situ hybridization using a probe to the woodchuck post-transcriptional regulatory element sequence included in the vector. These findings suggest that increased GABA production in lateral nucleus of the hypothalamus induced by GAD65 gene transfer may reduce weight gain through reduced feeding.

Molecular biology and gene therapy in the treatment of chronic pain.

Technologic advancements have made cell type-specific targeting, expression control, and safe and stable gene transfer possible. Animal research has provided increasing experience with gene transfer to the nervous system and sensory neurons in particular. Gene-based neuromodultion can be achieved through neuronal delivery of transgenes capable of altering synaptic function. Alternatively, ex vivo gene transfer can be used to create cell lines capable of secreting analgesic neurepeptides. Translatation of these grafts and direct gene-based neuromoduation can be applied to the control of pain and the root causes of pain. These approaches combine anatomic and pharmacologic specificity. As the technology continues to improve, clinical application of cellular and molecular pain control is likely.

Trophic activity of Rabies G protein-pseudotyped equine infectious anemia viral vector mediated IGF-I motor neuron gene transfer in vitro

The present study examines gene delivery to cultured motor neurons (MNs) with the Rabies G protein (RabG)-pseudotyped lentiviral equine infectious anemia virus (RabG.EIAV) vector. RabG.EIAV-mediated beta-galactosidase (RabG.EIAV-LacZ) gene expression in cultured MNs plateaus 120 h after infection. The rate and percent of gene expression observed are titer-dependent (P < 0.001). The rat IGF-I cDNA sequence was then cloned into a RabG.EIAV vector (RabG.EIAV-IGF-I) and was shown to induce IGF-I expression in HEK 293 cells. MNs infected with RabG.EIAV-IGF-I demonstrate enhanced survival compared to MNs infected with RabG.EIAV-LacZ virus (P < 0.01). In addition, IGF-I expression in cultured MNs induced profound MN axonal elongation compared to control virus (P < 0.01). The enhanced motor neuron tropism of RabG.EIAV previously demonstrated in vivo, together with the trophic effects of RabG.EIAV-IGF-I MN gene expression may lend this vector to therapeutic application in motor neuron disease.

Anatomically discrete functional effects of adenoviral clostridial light chain gene-based synaptic inhibition in the midbrain

The gene for the Light Chain fragment of Tetanus Toxin (LC) induces synaptic inhibition by preventing the release of synaptic vesicles. The present experiment applied this approach within the rat midbrain in order to demonstrate that LC gene expression can achieve functionally and anatomically discrete effects within a sensitive brain structure. The deep layers of the superior colliculus/deep mesencephalic nucleus (dSC/DpMe) that are located in the rostral midbrain has been implicated in fear-induced increase of the acoustic startle reflex (fear potentiated startle) but exists in close proximity to neural structures important for a variety of critical functions. The dSC/DpMe of adult rats was injected bilaterally with adenoviral vectors for LC, green fluorescent protein, or vehicle. Synaptobrevin was depleted in brain regions of adenoviral LC expression. LC gene expression in the dSC/DpMe inhibited the increase in startle amplitude seen with the control viral infection, and blocked context-dependent potentiation of startle induced by fear conditioning. Although LC gene expression reduced the absolute amount of cue-specific fear potentiated startle, it did not decrease percent potentiated startle to a cue, nor did it reduce fear-induced contextual freezing, nonspecific locomotor activity, or general health, indicating that its effects were functionally and anatomically specific. Thus, vector-driven LC expression inhibits the function of deep brain nuclei without altering the function of surrounding structures supporting its application to therapeutic neuromodulation.

240. Hemiparkisonism Induced through Unilateral Expression of Adenovirus-Mediated Clostridial Light Chain Gene in the Substantia Nigra

Background: Clostridial light chain (LC) inhibits synaptic transmission by digesting a vesicle-docking protein, synaptobrevin, without altering neuronal health. We have previously reported focal synaptic inhibition through adenovirus-mediated gene transfer of LC (AdLC) in the central nervous system. We report here the feasibility of creating reversible rat hemiparkinsonism (HPD) model through AdLC injection into the substantia nigra (SN).