The effects of promoter specificity on transgene expression in the mouse spinal cord after remote delivery of Rabies-pseudotyped HIV-1-based lentiviral vector
¹Eric Quach, Brooke Snyder, Thais Federici, and Nicholas M. Boulis
¹Department of Neurosurgery, Emory University, Atlanta, GA

Abstract

Although many vector systems with varying attributes currently exist for gene-based therapies, viral vectors exhibiting a neuronal tropism coupled with the ability to undergo retrograde axonal transport may be most appropriate for use in treatment of motor neuron diseases. The Boulis Lab previously determined that human immunodeficiency virus type 1 (HIV-1)-based lentiviral (LV) vector pseudotyped with rabies PV G-glycoprotein (RabPVG) is an ideal vector for retrograde transport. In this study, we further assessed retrograde delivery of the RabPVG HIV-1-based LV vector in vivo with unilateral injections into the mouse medial gastrocnemius muscle. GFP expression, driven by either the constitutive hybrid cytomegalovirus (CMV) enhancer/chicken β-actin (CAG) promoter or the motor neuron-specific 3.6-kb Hb9 promoter, was examined in the spinal cord. In vitro the CAG promoter yielded superior GFP expression in SH-SY5Y cells and mixed spinal cord cultures, whereas the 3.6-kb Hb9 promoter resulted in higher GFP expression in the spinal cord in vivo. These results suggest that promoter specificity, even with vector delivered in the periphery, may impact transgene expression in the CNS.

Introduction

Lentiviral (LV) Vectors

Subfamily of retroviruses capable of transducing both dividing and nondividing cells like motor neurons

Envelope pseudotyping of the glycoproteins alters tropism of the virus as well as its retrograde transport ability

Larger packaging capacity than other viral vectors

HIV-1-based LV vector pseudotyped with rabies PV G-glycoprotein (RabPVG) has been shown to readily undergo retrograde transport and exhibits a neuronal tropism

Remote Gene Delivery

Intramuscular (IM) and intraneural injections

Allows for transduction of neuronal cell bodies localized in regions distant from the injection site via retrograde transport of virus

Less invasive than direct injection methods

Promoters

Can restrict gene expression to certain cell types for targeted therapies

CMV enhancer/chicken β-actin (CAG) promoter - comprised of the chicken β-actin promoter downstream of the CMV enhancer

3.6-kb Hb9 (Hb9) promoter – Hb9 is a homeobox gene that is selectively expressed in both developing and adult motor neurons

Methods and Materials

In vitro transduction

Human neuroblastoma SH-SY5Y cells and mixed spinal cord cultures were incubated overnight with RabPVG – CAG or RabPVG – Hb9 LV vectors encoding GFP. Pictures taken at 48 hrs. Multiplicty of infection (MOI) = 10.



Injection procedure repeated for RabPVG – Hb9 LV vector (n = 3)

Immunohistochemistry and Tissue Analysis

Spinal cord and muscle specimens were cryosectioned and stained for GFP

CTB labeled cells and GFP positive cells were counted

Results

Conclusions and Future Studies

Promoter specificity may play a role in increasing vector efficiency when targeting specific populations of cells

Using a cell-specific promoter does not adversely affect transgene expression mediated by a peripherally injected vector

Future Directions

Evaluate the effects of the CAG and Hb9 promoters on transgene expression in other delivery methods such as intraparenchymal injections

Examine RabPVG HIV-1-based LV vector delivery of potentially therapeutic genes such as SMN1 or growth factor genes such as IGF-1 in a spinal muscular atrophy (SMA) mouse model

Resources

This material is based upon work supported by the Howard Hughes Medical Institute under Grant No. 52005873 and by the Muscular Dystrophy Association.

References

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Federici, T., R. Kutner, et al. (2009). "Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuronal gene transfer." Genetic Vaccines and Therapy 7(1): 1.

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Mazarakis, N., M. Azzouz, et al. (2001). "Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery." Hum Mol Genet 10: 2109 - 2121.

Thais Federici and Nicholas M. Boulis (2006). "Gene-based treatment of motor neuron diseases." Muscle & Nerve 33(3): 302-323.