Qingshan Teng

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.

Intraspinal cord delivery of IGF-I mediated by adeno-associated virus 2 is neuroprotective in a rat model of familial ALS.

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a devastating disease that is characterized by the progressive loss of motor neurons. Patients with ALS usually die from respiratory failure due to respiratory muscle paralysis. Consequently, therapies aimed at preserving segmental function of the respiratory motor neurons could extend life for these patients. Insulin-like growth factor-I (IGF-I) is known to be a potent survival factor for motor neurons. In this study we induced high levels of IGF-I expression in the cervical spinal cord of hSOD1(G93A) rats with intraspinal cord (ISC) injections of an adeno-associated virus serotype 2 vector (CERE-130). This approach reduced the extent of motor neuron loss in the treated segments of the spinal cord. However, a corresponding preservation of motor function was observed in male, but not female, hSOD1(G93A) rats. We conclude that ISC injection of CERE-130 has the potential to protect motor neurons and preserve neuromuscular function in ALS.

Cervical Spinal Cord Therapeutics Delivery: Preclinical Safety Validation of a Stabilized Microinjection Platform

OBJECTIVEThe current series represents a preclinical safety validation study for direct parenchymal microinjection of cellular grafts into the ventral horn of the porcine cervical spinal cord. METHODSTwenty-four 30- to 40-kg female Yorkshire farm pigs immunosuppressed with cyclosporine underwent a cervical laminectomy and ventral horn human neural progenitor cell injection. Cell transplantation in groups 1 to 3 (n = 6 pigs each) was undertaken with the intent of assessing the safety of varied injection volumes: 10, 25, and 50 μL injected at 1, 2.5, and 5 μL/min, respectively. Groups 4 and 5 (n = 3 pigs each) received prolonged immunosuppressant pretreatment in an attempt to demonstrate graft viability. The latter was undertaken in an alternate species (mini-pig versus Yorkshire pig). RESULTSNeurological morbidity was observed in 1 animal and was attributable to the presence of a resolving epidural hematoma noted at necropsy. Although instances of ventral horn targeting were achieved in all injection groups with a coordinate-based approach, opportunities exist for improvement in accuracy and precision. A relationship between injection volume and graft site cross-sectional area suggested limited reflux. Only animals from group 5 achieved graft survival at a survival end point (t = 1 week). CONCLUSIONThis series demonstrated the functional safety of targeted ventral horn microinjection despite evidence for graft site immune rejection. Improvements in graft delivery may be augmented with an adapter to improve control of the cannula entry angle, intraoperative imaging, or larger graft volumes. Finally, demonstration of long-term graft viability in future preclinical toxicity studies may require tailored immunosuppressive therapies, an allograft construct, or tailored choice of host species.

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.

A means for targeting therapeutics to peripheral nervous system neurons with axonal damage

OBJECTIVEDelivery of biological therapeutics to motor and dorsal root ganglion neurons remains a major hurdle in the development of treatments for a variety of neurological processes, including peripheral nerve injury, pain, and motor neuron diseases. Because nerve cell bodies are important in initiating and controlling axonal regeneration, targeted delivery is an appealing strategy to deliver therapeutic proteins after peripheral nerve injury. METHODSTet1 is a 12-aa peptide, isolated through phage display that is selected for tetanus toxin C fragment-like binding properties. In this study, we surveyed its uptake and retrograde transport using compartmented cultures and sciatic nerve injections. We then characterized the time course of this delivery. Finally, to confirm the retrograde transport involvement, a colchicine pretreatment was performed. We also performed competitive binding studies between Tet1 and a recombinant tetanus toxin C fragment using recombinant tetanus toxin C fragment enzyme-linked immunosorbent assay. RESULTSWe were able to demonstrate efficient uptake and retrograde axonal transport of the Tet1 peptide in vitro and in vivo. Intraneural colchicine pretreatment partially blocked fluorescence detection in the spinal cord, revealing a retrograde axonal transport mechanism. Finally, a competitive enzyme-linked immunosorbent assay experiment revealed Tet1-specific binding to the recombinant tetanus toxin C fragment axon terminal trisialogangliosides receptor. CONCLUSIONThese properties of Tet1 can be applied to the development of therapeutic viral vectors and fusion proteins for neuronal targeting and enhanced spinal cord delivery in the treatment of nerve regeneration, neuroprotection, analgesia, and spasticity. Small peptides can be easily fused to larger proteins without significantly modifying their function and can be used to alter the binding and uptake properties of these proteins.

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

Fusion of the tetanus toxin C fragment binding domain and Bcl-xL for protection of peripheral nerve neurons.

OBJECTIVE Apoptosis has been shown to play an important role in motor neuron (MN) degeneration in both neurodegenerative disease and peripheral neuropathy. Bcl-xL, an antiapoptotic protein, is down-regulated in these etiologies. The carboxyl-terminal domain of the tetanus toxin heavy chain (Hc) has high affinity for axon terminal binding and uptake into motor and dorsal root ganglion (DRG) neurons. We report the development of a fusion protein between Hc and Bcl-xL to enhance uptake of Bcl-xL by MNs as a strategy for inhibiting peripheral neuronal apoptosis. METHODS The genes for Hc, Bcl-xL, and green fluorescent protein were cloned into an Escherichia coli expression system in 2 different arrangements. Fusion proteins were purified through chromatography. Cultured E15 rat spinal cord MNs and DRG cells were used to demonstrate neuron-specific uptake and retrograde transport of the fusion proteins mediated by Hc. Finally, glutamate-induced apoptosis was used as an in vitro model to measure the antiapoptotic effects of the fusion proteins. RESULTS Bcl-xL fusion proteins were found to bind specifically and undergo uptake into cultured rat spinal MNs. The fusion proteins were also taken up by DRG axonal terminals and transported back to the cell bodies in Campenot compartmentalized chambers (Tyler Research Corp., Edmonton, Canada). Finally, fusion protein application improved cell survival and decreased apoptosis in glutamate-mediated excitotoxicity of the SH-SY5Y neuronal cells. CONCLUSION Hc can be applied as a universal carrier for therapeutic cargo delivery specifically to MNs or DRGs. The fusion proteins between Bcl-xL and Hc constructed in this study might bear applications to the treatment of MN disease, neuropathy, or nerve injury through nerve or intramuscular injection.

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.

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.