Methods and pharmaceutical compositions for expressing a polynucleotide of interest in the peripheral nervous system of a subject

Abstract

The present invention relates to methods and pharmaceutical compositions for expressing a polynucleotide of interest in the peripheral nervous system of a subject. In particular, the present invention relates to a method for selectively expressing a polynucleotide of interest in the peripheral nervous system of a subject in need thereof comprising the step of transducing a peripheral nerve of the subject with an amount of an AVV9 vector containing the polynucleotide of interest.

Claims

1. A method for expressing a polynucleotide in myelinating Schwann cells of a subject with a peripheral demyelinating disease, comprising injecting an AAV9 vector that encodes the polynucleotide into a peripheral nerve of the subject, wherein the AAV9 vector preferentially transduces the myelinating Schwann cells over non-myelinating Schwann cells along the peripheral nerve, and wherein the polynucleotide is expressed in transduced myelinating Schwann cells along the peripheral nerve.

2. The method of claim 1, wherein the peripheral demyelinating disease is selected from the group consisting of hereditary peripheral demyelinating diseases and acquired peripheral demyelinating diseases.

3. The method of claim 1, wherein the peripheral demyelinating disease is selected from the group consisting of Refsum's disease, Abetalipoproteinemia, Tangier disease, Krabbe's disease, Metachromatic leukodystrophy, Fabry's disease, Dejerine-Sottas syndrome, and Charcot-Marie-Tooth Diseases.

4. The method of claim 1, wherein the peripheral demyelinating disease is selected from the group consisting of diabetic neuropathies, immune-mediated neuropathies, acute motor neuropathy, acute sensory neuropathy, acute autonomic neuropathy, chronic polyneuropathies, peripheral demyelinating diseases associated with vasculitis or inflammation of the blood vessels in peripheral nerves, peripheral demyelinating diseases associated with monoclonal gammopathies, peripheral demyelinating diseases associated with tumors or neoplasms, peripheral demyelinating diseases caused by infections, peripheral demyelinating diseases caused by nutritional imbalance, peripheral demyelinating diseases arising in kidney diseases, hypothyroid neuropathies, peripheral demyelinating diseases caused by alcohol and toxins, peripheral demyelinating diseases caused by trauma or compression, and idiopathic peripheral demyelinating diseases.

5. The method of claim 1, wherein the polynucleotide is an antisense oligonucleotide construct.

6. The method of claim 1, wherein the polynucleotide is a siRNA.

7. The method of claim 1, wherein the polynucleotide is operatively linked to a promoter sequence.

8. The method of claim 1, wherein the AAV9 vector does not transduce axons.

9. The method of claim 1, wherein about 97% of the myelinating Schwann cells are transduced.

10. The method of claim 1, wherein the subject is a primate.

Description

FIGURES

(1) FIG. 1: Cryosection analysis on OCT-embedded adult mice sciatic nerves showing the transduction profile of AAV-injected sciatic nerves. Whole sciatic nerve is represented in the first lane, zoom at 20 is represented in the second lane.

(2) FIG. 2: Percentage of myelinating Schwann cells transduced among all the myelinating Schwann cells along the AAV9-injected PNH sciatic nerve. Analysis were performed on OCT and paraffin-embedded sciatic nerve sections.

EXAMPLE 1: INTRASCIATIC INJECTION ON ADULT MICE

(3) Methods:

(4) The methodology of injection is described in the following publication (Gonzalez et al., 2014) with the parameters below. Briefly, mice were anaesthetized with isoflurane, and were maintained under isoflurane during the surgery. Incision was made with a scalpel at the mid-thigh; sciatic nerve was lifted out with spatula and exposed. The vectors were co-injected into the sciatic nerve with Fast green (0.005% final concentration) via glass needles, at <45 acute angle to the nerve surface, connected to a micro injector linked to a pulse generator. The capillary remained in place at the injection site for 1 additional min, before it was slowly removed. The injection parameters are listed in the table below.

(5) TABLE-US-00001 TABLE 1 injection parameters on adult mice Vectors AAV9 and AAVrh10_CAG_eGFP Injection Unilateral in the right sciatic nerve Quantity of vector per nerve 4.6 10.sup.10 gc/nerve Volume injected per nerve 8 l Injection time 15 min Number of mice per vectors 9 Age of injection 2 months Sacrifice 1 month post injection

(6) Results

(7) To explore the ability of AAV vector to transduce axons or Schwann cells, we performed teasing analysis on injected sciatic nerves (three injected nerves per vector). Thus, among all the transduced cells, we determined the percentage of transduced myelinating Schwann cells, non-myelinating Schwann cells and axons (Table 2).

(8) TABLE-US-00002 TABLE 2 Teasing analysis of AAV-injected mice sciatic nerves showing the ability of AAV vector to transduce axons or Schwann cells. The results are presented as a percentage of axons or myelinating Schwann cells or non myelinating Schwann cells transduced among all the transduced cells. AAV9-CAG-eGFP AAV10-CAG-eGFP (N = 3) (N = 3) Mean SD Mean SD (%) (%) (%) (%) Myelinating Schwann cells 97 4 82 17 Non myelinating Schwann 3 3 4 4 cells Axons 0 0 14 13

(9) AAV9 transduced almost exclusively myelinating Schwann cells (97%), very few non myelinating Schwann (3%) cells and no axons. AAVrh10 shows a transduction profile slightly different with a strong proportion of myelinating Schwann cells (82%), very few non myelinating Schwann cells (4%) and some axons (14%)

(10) In parallel to teasing analysis, coronal sections of OCT-embedded sciatic nerves were performed (four injected sciatic nerves per vector). The profile of transduction obtained for each vector in a whole coronal section of the sciatic nerve at the injection site is described in FIG. 1. AAV9 clearly displayed a higher rate of transduction than AAVrh10. To determine the types of transduced cells, coronal sections of sciatic nerve were stained either for MBP, a myelinating Schwann cell marker, or for TUJ1, an axonal cell marker. We found that AAV9 and AAVrh10 expression were mostly detected in myelinating Schwann cells. At the injection site, up to 93% of positive myelinating Schwann cells were counted with AAV9, whereas only 51% with AAVrh10. Moreover, along the sciatic nerve, AAV9 displayed a better diffusion than AAVrh10, with respectively 63 and 42% at 1 cm proximal from the injection site, 91 and 42% at 1 cm distal from the injection site (Table 3).

(11) TABLE-US-00003 TABLE 3 Percentage of myelinating Schwann cells transduced among all the myelinating Schwann cells along the AAV-injected mice sciatic nerves. AAV9-CAG-eGFP AAV10-CAG-eGFP (N = 3) (N = 3) Mean SD Mean SD (%) (%) (%) (%) 1 cm proximal from 63 24 42 22 the Injection site Injection site 93 2 51 11 1 cm distal from 91 2 42 16 the injection site

(12) Moreover, vector genome copy (VGC) from entire sciatic nerve for each group of injected adult mice (three mice per vector) was evaluated. First, DNA extraction was performed thanks to DNA blood and tissue kit (Qiagen) by following the manufacturer instructions. Then, qPCR was realized using two different primers, one against eGFP transgene and one against ITR sequence. The results are presented in table 4.

(13) TABLE-US-00004 TABLE 4 VGC evaluated by qPCR on DNA extracted from the entire sciatic nerve using either primers against eGFP transgene or primers against ITR sequence. AAV9-CAG-eGFP AAV10-CAG-eGFP (N = 3) (N = 3) Mean SD Mean SD VGC eGFP 0.551 0.146 0.525 0.084 ITR 0.507 0.077 0.338 0.113

(14) The results showed the ability of AAV vector to transduce mouse sciatic nerve with roughly a similar pattern for AAV9 and AAVrh10 with VGC around 0.5.

EXAMPLE 2: INTRASCIATIC INJECTION ON PUPS MICE (P3-P4)

(15) Methods:

(16) The same methodology of injection described in the example 1 was used with the following parameters (Table 5):

(17) TABLE-US-00005 TABLE 5 injection parameters in pups' mice Vectors AAV9-CAG-eGFP Injection Unilateral in the right sciatic nerve Quantity of vector per nerve 1.15 l0.sup.10 gc/nerve Volume injected per nerve 2 l Injection time 3-4 min Number of mice per vectors 7 Age of injection P3-P4 Sacrifice 1 month post injection

(18) Results

(19) To explore the ability of AAV vector to transduce axons or Schwann cells, we performed teasing analysis on injected sciatic nerves (three pups' mice). Teasing results are presented in the Table 6.

(20) TABLE-US-00006 TABLE 6 Teasing analysis of AAV-injected mice sciatic nerves showing the ability of AAV vector to transduce axons or Schwann cells. The results are presented as a percentage of axons or myelinating Schwann cells or non-myelinating Schwann cells transduced among all the transduced cells. AAV9-CAG-eGFP (N = 3) Mean (%) SD (%) Myelinating Schwann cells 87 12 Non myelinating Schwann cells 2 2 Axons 14 12

(21) In pups mice, AAV9 transduced almost exclusively myelinating Schwann cells (87%), few non myelinating Schwann (14%) cells and very few axons (2%).

(22) In parallel to teasing analysis, coronal sections of OCT-embedded sciatic nerves were performed (three injected mice), as described in the example 1. On the injection site, up to 84% of positive myelinating Schwann cells were counted. At 1 cm proximal from the injection site, 74% of myelinating Schwann cells were transduced and at 1 cm distal from the injection site, 74% of myelinating Schwann cells were transduced (Table 7).

(23) TABLE-US-00007 TABLE 7 Percentage of myelinating Schwann cells transduced among all the myelinating Schwann cells along the AAV-injected pups' mice sciatic nerves. AAV9-CAG-eGFP (N = 3) Mean (%) SD (%) 1 cm proximal from the Injection site 74 7 Injection site 85 15 1 cm distal from the injection site 74 14

EXAMPLE 3: INTRASCIATIC INJECTION ON RATS

(24) Methods:

(25) The same methodology of injection described before was used with the following parameters (Table 8):

(26) TABLE-US-00008 TABLE 8 injection parameters in rats Vectors AAV9_CAG_eGFP Injection Unilateral in the right sciatic nerve Quantity of vector per nerve 1.8 10.sup.11 gc/nerve Volume injected per nerve 30 l Injection time 15 min Number of mice per vectors 7 Age of injection 1 month Sacrifice 1 month post injection

(27) Results

(28) Teasing analysis (three injected rats) and coronal sections of OCT-embedded sciatic nerves (three injected rats) were performed. The results are presented below (Table 9).

(29) TABLE-US-00009 TABLE 9 Teasing analysis of AAV-injected mice sciatic nerves showing the ability of AAV vector to transduce axons or Schwann cells. The results are presented as a percentage of axons or myelinating Schwann cells or non- myelinating Schwann cells transduced among all the transduced cells. AAV9-CAG-eGFP (N = 3) Mean (%) SD (%) Myelinating Schwann cells 89 9 Non myelinating Schwann cells 7 5 Axons 4 4

(30) In rats, AAV9 transduced almost exclusively myelinating Schwann cells (89%), very few non myelinating Schwann (7%) cells and very few axons (4%).

(31) In parallel to teasing analysis, coronal sections of OCT-embedded sciatic nerves were performed (three injected rats), as described before. Along all the nerve, up to 80% of myelinating Schwann cells were transduced (Table 10).

(32) TABLE-US-00010 TABLE 10 Percentage of myelinating Schwann cells transduced among all the myelinating Schwann cells along the AAV-injected rat sciatic nerves. AAV9-CAG-eGFP (N = 3) Mean (%) SD (%) Among all the nerve 80 14

EXAMPLE 4: INTRASCIATIC INJECTION ON NON-HUMAN PRIMATE (NHP)

(33) Methods

(34) Anaesthetized NHP are placed in a ventral decubitus prone position. After sciatic nerve exposure, the vector was co-injected with fast green (0.005% final concentration) into the left tibial nerve lcm above the bifurcation between the common fibular nerve and the tibial nerve. First, a 22 gauge needle is inserted into the epineurium (4 mm) and then a silica capillary, containing the vector and connected to a micropump, is pulled up through the needle. The capillary remained in place on the injection site for 1-2 additional min, before it was slowly removed. The injection parameters are listed in the table below (Table 11).

(35) TABLE-US-00011 TABLE 11 Injection parameters in non-human primate Vectors AAV9_CAG_eGFP AAVrh10_CAG_eGFP Injection Unilateral in the left sciatic nerve Quantity of vector 5 10.sup.12 gc/nerve 5 10.sup.12 gc/nerve per nerve Volume injected 400 l 400 l per nerve Injection time 30 min 30 min Number of NHP 1 1 per vectors Age of injection 2 years 2 years Sacrifice 1 month post 1 month post injection injection

(36) Results:

(37) Similarly to the AAV-injected mice, we analyzed the profile of transduction and the types of transduced cells. Immunostaining on coronal sections of OCT-embedded sciatic nerve showed that AAV9-driven expression was seen exclusively in myelinating Schwann cells and not in axons (FIG. 2). On NHP, only myelinating Schwann cells are transduced and no axons are transduced. The AAV9 GFP expression was detected along the sciatic nerve in a total distance of 5.5 cm, more precisely 3.5 cm proximal from the injection site and 1.5 cm distal from the injection with respectively 20 and 70% of transduced myelinating Schwann cells (FIG. 2). Up to 70% of positive myelinating Schwann cells were found on the injection site. This percentage decreased with the proximal distance from the injection site and was constant up to 1.5 cm distal from the injection site (FIG. 2). These results were confirmed by paraffin-embedded sciatic nerve sections

CONCLUSION

(38) Intrasciatic injection of AAV9 on mice (pups and adults), rats and non-human primate showed a strong transduction rate of myelinating Schwann cells with a good diffusion of the vector, never obtained and described in the literature up to now. These results highlight that AAV9 vector could represent a useful therapeutic tool to express proteins mutated or deregulated in myelinating Schwann cells in-pathologies affecting the peripheral nervous system.

REFERENCES

(39) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.