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Widespread gene delivery to motor neurons using peripheral injection of aav vectors

a technology of aav and aav, which is applied in the direction of muscular disorders, immunological disorders, drug compositions, etc., can solve the problems of failure of classical pharmacology, difficult alternative supply of mn with recombinant proteins injected directly into the cns parenchyma, and no treatment for these diseases

Pending Publication Date: 2018-03-08
GENETHON +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0070]Further aspects and advantages of the present inventions will be disclosed in the following experimental section, which shall be considered as illustrative only, and not limiting the scope of this application.

Problems solved by technology

There is no treatment for these diseases, mostly because drug delivery to MN via systemic injections is hindered by the presence of the “blood-brain-barrier” (BBB).
The alternative supply of MN with recombinant proteins injected directly into the CNS parenchyma is also difficult due to the invasiveness of the surgical procedure hampering a potential clinical application.
Failure of the classical pharmacology has led the scientific community to develop new therapeutic strategies based, in particular, on gene transfer technology using viral vectors.
However, these invasive approaches failed to produce efficient widespread CNS transduction.
However, diffusion of the recombinant proteins to the whole nervous tissue was far from being optimal, and again, the potential risks related to the surgical procedure is an obstacle to the clinical application of this method.
However, the clinical value of this method remains questionable due, in particular, to the large number of both injection sites and viral particles that would be needed for targeting MN in pathologies that affect most of the patient's motor units.

Method used

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  • Widespread gene delivery to motor neurons using peripheral injection of aav vectors
  • Widespread gene delivery to motor neurons using peripheral injection of aav vectors
  • Widespread gene delivery to motor neurons using peripheral injection of aav vectors

Examples

Experimental program
Comparison scheme
Effect test

example 1

ular Injection of mSEAP-Expressing AAV Vectors in the Neonatal Mouse

[0090]We first evaluated the potential of serotype 1 and 9 ss- or scAAV vectors to transduce the CNS cells after i.m. injection. The ssAAV1, ssAAV9, scAAV1 or scAAV9 encoding the murine secreted alkaline phosphatase (mSEAP) under the cytomegalovirus (CMV) promoter were injected into both triceps and gastrocnemius muscles in one day old C57Bl6 mice (8.10+9 to 2.10+10 viral genome per mouse, 3 mice per group). The injected muscle, brain and spinal cord tissues were removed 1, 3 or 7 days post injection and analysed for mSEAP expression using histochemistry.

[0091]The mSEAP expression was detected in the injected muscles 3 and 7 days after injection of each AAV serotype, except with ssAAV9, the expressing level dramatically increasing with time (FIG. 1a). In the CNS, transgene expression was detected only after i.m. injection of scAAV9. Interestingly, the mSEAP expression was detected in the epithelial cells of the chor...

example 2

toneal Injection of mSEAP-Expressing AAV Vectors in the Neonatal Mouse

[0092]We then analysed whether i.p. administration of ssAAV1, ssAAV9, scAAV1 and scAAV9 in one day old C57Bl6 mice (100 μl, 3·10+10 to 1·10+11 viral genome per mouse) could mediate transgene expression in the CNS at 1, 3 or 7 days post injection.

[0093]A low level of mSEAP expression was detected in the diaphragm muscle fibres of mice injected with ssAAV1 by 3 days post-injection, which was similar to that observed with scAAV1 (FIG. 2a), and with both vectors at 7 days after injection. (FIG. 2a). SsAAV9 transduce a few muscle fibres only at 21 days post injection, whereas an intense mSEAP staining was found in the diaphragm when using scAAV9 from 3 days post-injection (FIG. 2a). This high level of transduction was also observed in other muscles such as the triceps brachii or gastrocnemius muscle (data not shown).

[0094]The epithelial cells of the choroids plexus and the ependyma appeared clearly labelled after injec...

example 3

Expression in the CNS after i.m. Or i.p. Injection of scAAV9-GFP in the Neonatal Mouse

[0095]Since mSEAP is a secreted protein, the transgene expression observed in the CNS cells after peripheral AAV injection could result from protein transcytosis rather than from AAV cell transduction. We thus verified whether similar results could be obtained when using a non-secreted protein.

[0096]A recombinant scAAV9 vector expressing the “green fluorescent protein” (GFP) was injected in neonatal mice either intraperitoneally (3.10+10 vg per mouse, 100 μl) or intramuscularly (8.10+9 vg in 20 μl per mouse, 5 μl per muscle). Seven days later, and similarly to that observed with scAAV9-mSEAP, the GFP expression was observed in the choroids plexus and ependyma cells located in the brain ventricles (FIG. 3a and FIG. 4a). Furthermore, we found in this case many GFP-positive neural cells in several brain regions located, in particular, close to the ventricles. Cell bodies and fibres in the hippocampus ...

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Abstract

The present invention relates to compositions and methods, in particular to methods based on systemic injection of rAAV, for delivering genes to cells of the central nervous system in mammals, such as brain neurons or glial cells, and in particular to motor neurons or glial cells of the spinal cord The invention also relates to methods of treating motor neuron disorders in mammals by expression of therapeutic genes. The invention stems from the unexpected discovery that peripheral injection of AAV vectors leads to a bypass of the blood brain barrier and a massive infection of motor neurons. The invention may be used in any mammal, including human subjects.

Description

[0001]The present invention relates to compositions and methods for delivering genes to cells of the central nervous system in mammals. The invention also relates to methods of treating motor neuron disorders in mammals by expression of therapeutic genes. The invention stems from the unexpected discovery that peripheral injection of AAV vectors leads to a bypass of the blood brain barrier and a massive infection of motor neurons, as well as other cells in the central nervous system. The invention may be used in any mammal, including human subjects.INTRODUCTION[0002]Motor neuron (MN) diseases, such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) or Kennedy's disease, are neurodegenerative disorders characterised by the selective degeneration of MNs in the spinal cord, brainstem and / or motor cortex (Monani 2005; Pasinelli and Brown 2006); (MacLean, Warne et al. 1996). There is no treatment for these diseases, mostly because drug delivery to MN via systemic inject...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86
CPCC12N15/86A61K48/00C12N2750/14143C12N2830/008A61P17/02A61P21/00A61P25/00A61P25/04A61P25/20A61P25/28A61P29/00A61P35/00A61P37/00A61P37/02A61P37/06A61P43/00A61K48/0083A61K48/005
Inventor BARKATS, MARTINE
Owner GENETHON
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