VIRAL VECTOR TRANSDUCTION

Abstract

The invention relates to a method of improving the efficiency of transduction of viral vectors into cells, wherein the method comprises administering to a cell an antimalarial agent and a viral vector. The invention also relates to a composition comprising an antimalarial agent and a viral vector for use in increasing the efficiency of transduction of the viral vectors. The invention further relates to use of the method and compositions of the invention to treat a disease or disorder.

Claims

1. A method of improving the efficiency of transduction of viral vectors into cells, wherein the method comprises administering to a cell an antimalarial agent and a viral vector.

2. The method of claim 1 wherein the antimalarial agent and viral vector are administered simultaneously, sequentially or separately.

3. The method of claim 1 or claim 2 wherein the antimalarial agent is selected from the group comprising or consisting of 4-aminoquinolines (such as hydroxychloroquine, chloroquine and amodiaquine), 8-aminoquinolines (such as primaquine, pamaquine and tafenoquine), mefloquine, quinine, mepacrine, atovaquone, doxycycline, and a salt or derivative thereof.

4. The method of any preceding claim wherein the antimalarial agent is a quinoline compound.

5. The method of claim 4 wherein the antimalarial agent is selected from the group comprising or consisting of hydroxychloroquine, chloroquine, mefloquine, amodiaquine, quinine, pamaquine, primaquine, mepacrine, and a salt, or a derivative thereof.

6. The method of claim 5 wherein the antimalarial agent is hydroxychloroquine.

7. The method of any preceding claim wherein the viral vector is selected from the group comprising or consisting of an adeno-associated virus (AAV), an adenovirus, a retrovirus, a lentivirus, a vaccinia/poxvirus, or a herpesvirus.

8. The method of claim 7 wherein the viral vector is an adeno-associated viral (AAV) vector.

9. The method of any preceding claim wherein the antimalarial agent is hydroxychloroquine and the viral vector is an adeno-associated viral (AAV) vector.

10. The method of any preceding invention wherein the method is carried out in vitro or in vivo.

11. The method of any preceding claim wherein the antimalarial agent and/or viral vector are administered systemically or locally; and/or wherein the antimalarial agent and the viral vector are administered to the same cells, tissue or organ; and/or wherein the antimalarial agent and the viral vector are co-administered; and/or wherein the antimalarial agent and/or viral vector are administered at the intended site of transduction.

12. The method of any preceding claim wherein the viral vectors carry a cargo which is expressed upon transduction.

13. The method of any preceding claim wherein more than one viral vector is administered.

14. The method of claim 12 wherein the cargo comprises a transgene or part thereof intended to treat a disease or disorder; or wherein the cargo comprises at least one component needed to facilitate CRISPR gene editing; or wherein the cargo comprises an inhibitory RNA or a Mirtron; or wherein the cargo comprises one or more components needed to deliver an optogenetic therapy or system to a cell, tissue or organ.

15. A method of any of claims 1 to 9 wherein the method is used to increase the yield of recombinant AAV particles during a production run of the viral vector.

16. An antimalarial for use in increasing the transduction efficiency of a viral vector into a cell.

17. The antimalarial for the use of claim 16 wherein the antimalarial agent is hydroxychloroquine and the viral vector is an adeno-associated viral (AAV) vector.

18. A composition comprising a viral vector and an antimalarial agent.

19. A pharmaceutical composition comprising a viral vector, an antimalarial agent, and a pharmaceutically acceptable carrier, diluent or excipient.

20. A pharmaceutical composition according to claim 34 wherein the composition comprises an AAV vector, and one or more of hydroxychloroquine, chloroquine, and mefloquine.

21. A pharmaceutical composition according to claim 19 or 20 wherein the composition is intended for ocular administration.

22. The composition or pharmaceutical composition according to any of claims 18 to 21, the use of claim 16 or 17, or the method of any of claims 1 to 15, for use in the treatment of a disease or disorder.

23. The composition or pharmaceutical composition, or use, or method, according to claim 22 wherein the disease or disorder is a disease or disorder of the eye; or where the composition, use, or method, is to enhance the gene therapy treatment of an ocular disorder or disease, and optionally wherein the viral vector and/or antimalarial agent are administered directly to the eye, for example by subretinal, suprachoroidal, intravitreal, peri-ocular or anterior chamber injection.

24. The composition or pharmaceutical composition, or use, or method, according to claim 22 wherein the disease or disorder is a CNS condition, and optionally wherein the AAV vector and/or the antimalarial agent are administered by direct spinal cord injection and/or intracerebral administration.

25. The composition or pharmaceutical composition, or use, or method, according to claim 22 wherein the proportion of cells transduced by the viral vector in a target cell population is increased by at least 1% in the presence of the antimalarial agent compared to using the viral vector alone; or wherein the proportion of cells transduced by the viral vector in a target cell population is increased by at least 1 fold in the presence of the antimalarial agent compared to using the viral vector alone.

26. The composition or pharmaceutical composition according to any of claims 18 to 21, the use of claim 16 or 17, or the method of any of claims 1 to 15 wherein the antimalarial agent is used at a concentration of between about 1 M and about 30 M.

27. A method of treating a disease or disorder in a subject, wherein the method comprises administering to the subject a pharmaceutical composition according to claim 19 or 20, optionally the disease or disorder is a disease or disorder of the eye or is a CNS condition.

28. A kit for use in increasing the efficiency of transduction of a viral vector, wherein the kit comprises a viral vector to be transduced and an antimalarial agent.

29. The kit of claim 28 wherein the viral vector and antimalarial agent are provided in the same composition.

30. The kit of claim 41 wherein the viral vector is provided in a first container and the antimalarial agent is provided in a second container, and optionally further comprising instructions to mix the contents of the first and second containers prior to administration, and/or further comprising instructions when to administer the antimalarial agent, and when to administer the viral vector.

31. The kit of any of claims 28 to 30 further comprising one or more syringes for use in injecting the viral vector and/or the antimalarial agent.

Description

[0066] The present invention will be further described in more detail, by way of example only, with reference to the following figures in which:

[0067] FIGS. 1a and 1b illustrate the effect of hydroxychloroquine (HCQ) on AAV transduction in mouse embryonic fibroblasts (MEFs) and demonstrate the effect to be dose dependent.

[0068] FIGS. 2a, 2b and 2c illustrate the effect of hydroxychloroquine (HCQ) on AAV transduction in non-human primate (NHP) retinal pigment epithelium (RPE). The NHP RPE was obtained from Rhesus macaque (M. mullata) eyes obtained from the MRC Centre for Macaques (Porton Down, UK)

[0069] FIGS. 3a and 3b illustrate the effect to hydroxychloroquine (HCQ) on AAV transduction in human retinal explants.

[0070] FIGS. 4a, 4b and 4c illustrate that hydroxychloroquine (HCQ) improves the efficacy of AAV transduction in vivo following co-administration of HCQ and an AAV vector encoding green fluorescent protein (GFP) into the subretinal space in mice.

[0071] FIGS. 5a and 5b illustrate the effect of chloroquine (CQ) on AAV transduction and demonstrate the effect to be dose dependent.

EXAMPLE 1

[0072] Demonstrates that hydroxychloroquine (HCQ) improves the efficacy of AAV transduction in a dose dependent manner.

[0073] Methods:

[0074] Cell culture. Wildtype (WT) mouse embryonic fibroblasts (MEFs) were seeded into 6-well plates. 24 hr after seeding the cells were incubated in culture media with HCQ concentrations of 3.125-50 uM for 1 hr prior to transduction with a rAAV2.CAG.GFP.WPRE.pA vector at a MOI of 1000. The cells were cultured in HCQ containing media until harvesting. Cells were imaged 3 days post-transduction (FIG. 1A).

[0075] Flow cytometry analysis. Cells were processed 3 days post-transduction and stained with the cell viability dye 7-AAD for 5 minutes prior to flow cytometry analysis. Fluorescent light was measured by a BD LSRFortessa flow cytometer using a blue laser with the bandpass filters 695 nm/40 mW and 530 nm/30 mW to detect the fluorochromes of 7-AAD and GFP, respectively (FIG. 1a).

[0076] Results & Discussion: [0077] (a) The results presented in FIGS. 1a and 1b demonstrate that increasing concentrations of HCQ increase the number of GFP expressing cells up to 18.75 uM. At concentrations of 25 uM and above HCQ induces cell death which in turn decreases the proportion of live GFP expressing cells. [0078] (b) The results also demonstrate that HCQ increases the number of GFP expressing cells in a dose dependent manner in WT MEFs.

[0079] The results presented demonstrate that the antimalarial agent HCQ improves the efficacy of AAV transduction.

EXAMPLE 2

[0080] Demonstrates that HCQ improves the efficacy of AAV transduction in non-human primate (NHP) retinal pigment epithelium (RPE) cells

[0081] Methods:

[0082] Harvesting non-human primate (NHP) retinal pigment epithelium (RPE) cells. After enucleation, the cornea and lens were removed under direct visualization with a surgical microscope. Radial incisions were made towards the posterior pole to flatten the eyecup. The retina was removed by blunt dissection and the remaining eyecup was placed in media and stored on ice until the RPE cells were removed. RPE cells were detached using the TrypLE cell dissociation reagent and were cultured at 37 C.

[0083] Cell culture. Primary NHP RPE cells were seeded into a 12-well plate. 24 hr after seeding, the cells were incubated in media with 3.125 uM or 18.75 uM of HCQ for 1 hr prior to transduction with 210.sup.9 genome copies per well. Cells were imaged 3 days post-transduction.

[0084] RNA & protein extraction and cDNA synthesis. The primary NHP RPE were harvested for RNA extraction 3-days post-AAV transduction with HCQ. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Copy DNA was synthesised using 850-1500 ng of RNA using the Superscript III Kit (Invitrogen) with an oligo dT primer.

[0085] qPCR analysis. qPCR was carried out using TaqMan probes against GFP and -actin in a CFX Connect thermal cycler (BioRad). GFP expression was normalized to -actin and represented as a fold change to transduced cells incubated without HCQ.

[0086] Protein extraction. The flow through from RNA extraction above was combined with 4 volumes of acetone and incubated on ice for 30 min. Samples were centrifuged at 17,000g for 30 min, the supernatant was discarded and the pellet washed in 100 L of ice-cold ethanol. Pellets were resuspended in 1% SDS.

[0087] Western blot. Total protein content in cell lysates was determined using the bicinchoninic acid method. GFP and actin proteins were probed using monoclonal antibodies, and detected using ECL and Odyssey Imaging System (LI-COR). Data analysis was performed using ImageStudio Lite software (LI-COR). GFP band density was normalised to -actin.

[0088] Results & Discussion:

[0089] GFP mRNA expression was increased by 3 fold (N=3, p=0.0253) and GFP protein expression increased by 19 fold at 3 days post-transduction when primary NHP RPE cells were treated with 18.75 M of HCQ compared to transduced cells incubated without HCQ (FIGS. 2a and 2b). These results demonstrate that 18.75 M of HCQ improved AAV transduction in the primary NHP RPE cells.

[0090] Protein was extracted from the RNA extraction flow through, however this yielded low concentrations so it was only possible to run a western blot on N=2, which showed that 18.75 M of HCQ improved GFP protein expression by approximately 4-fold in the NHP RPE cells (FIG. 2c).

EXAMPLE 3

[0091] Demonstrates that HCQ improves the efficacy of AAV transduction in human retinal explants.

[0092] Methods:

[0093] Harvesting tissue. Human retina fragments was extracted during routine retinal surgery in which a retinectomy was required (ethics approved). Fragments of retina were removed via a 23G sclerostomy with vitrectomy cutter (at minimal cut rate) and manual aspiration.

[0094] Culturing explants. Within 1 hr of tissue collection, retinal fragments were transferred using a 3 mL Pasteur pipette into individual organotypic culture inserts (BD Falcon), which were in turn placed within a 24-well plate. 400 uL of media was placed beneath the insert and 100 uL of media within the insert. 24 hr after culturing the retinal explants, the media was replaced with media containing 3.125 uM or 18.75 uM of HCQ. The explants were incubated in the HCQ medium for 1 hour prior to transduction with 110.sup.9 genome copies of rAAV2.CAG.GFP.WPRE.pA vector. The explants were imaged every 2 days for 11 days and the mean grey value (representative of GFP fluorescence) of the explants was measured using ImageJ software (FIG. 3a).

[0095] Results & Discussion:

[0096] The AAV transduced explants demonstrated an up to 2-fold increase in mean grey value when treated with 3.125 uM HCQ compared to with no HCQ (n=6, p=0.0213) (FIG. 3b). This demonstrates that 3.125 M of HCQ improved AAV transduction.

EXAMPLE 4

[0097] Illustrate that hydroxychloroquine (HCQ) improves the efficacy of AAV transduction in vivo following co-administration of HCQ and an AAV vector encoding green fluorescent protein (GFP) into the subretinal space in mice.

[0098] Methods:

[0099] Subretinal injections. 7 week old female C57BL/6 mice were subretinally injected with 1.2 L vector suspension (110.sup.8 genome copies). The vector was prepared with a 3.125 uM or 18.75 uM final concentration of HCQ with control preparations without any HCQ. Animals were injected with an AAV+HCQ preparation in one eye and AAV only in the paired eye.

[0100] Confocal scanning laser ophthalmoscope (cSLO) analysis. Standardized mouse autofluorescence (AF) imaging using a cSLO (Spectralis HRA, Heidelberg Engineering) was performed in all animals according to previously published protocol. All images were recorded using the 55 lens of the Spectralis HRA at 2, 4 and 8 weeks post-injection. Images were recorded using a standardized signal detector sensitivity. For quantitative analysis of fundus AF, the mean grey level was measured within a ring-shaped area located at radii between 350 and 860 pixels from the optic disc centre using ImageJ software (NIH).

[0101] OCT analysis. Animals were subject to wide-field spectral-domain optical coherence tomography (OCT, Spectralis HRA, Heidelberg Engineering) at 2, 4 and 8 weeks post-injection. Mice were scanned using a 55 lens and 8 radial sections were taken with a real-time average process of 25 frames. The total retinal thickness and the outer limiting membrane (OLM) thickness were manually measured on alternate radial sections using a calliper.

[0102] Protein extraction. Mouse retinas were thawed on ice and lysed in 1 RIPA buffer+protease inhibitors. The retinas were homogenised using a hand-held homogeniser and incubated on ice for 30 mins. The samples were centrifuged at 17,000g at 4 C. for 20 mins. The supernatant was transferred to a new tube and kept on ice.

[0103] Western blot (WB). Total protein content in cell lysates was determined using the bicinchoninic acid method. GFP and actin proteins were probed using monoclonal antibodies, and detected using ECL and Odyssey Imaging System (LI-COR). Data analysis was performed using ImageStudio Lite software (LI-COR). GFP band density was normalised to actin.

[0104] Results & Discussion:

[0105] Quantification of the mean grey value of the fundus AF images demonstrated a statistically significant 2-fold increase in mean grey value (a surrogate measure of GFP expression) in the eyes subretinally injected with a suspension of AAV and 18.75 uM HCQ at 4 and 8 weeks post-injection compared to control eyes injected with AAV only (FIG. 4a).

[0106] There was no significant difference in the mean retinal thickness or retinal morphology between any of the groups with and without HCQ in the subretinal injection suspension (FIG. 4b). This suggests that subretinal HCQ did not have any detectable toxic effect on the retina of the animals.

[0107] FIG. 4c illustrates that there was an increase in the GFP signal detected by WB compared to the 0 M HCQ paired eye, demonstrating that 18.75 M HCQ increases GFP protein levels.

EXAMPLE 5

[0108] Demonstrates that chloroquine (CQ), like HCQ in Example 1, improves the efficacy of AAV transduction in a dose dependent manner.

[0109] Methods:

[0110] Cell culture. Wildtype (WT) mouse embryonic fibroblasts (MEFs) were seeded into 6-well plates. 24 hr after seeding, the cells were incubated in media with CQ concentrations of 1.5 to 12 uM for 1 hr prior to transduction with a rAAV2.CAG.GFP.WPRE.pA vector at a MOI of 1000. The cells were cultured in CQ containing media until harvesting. Cells were imaged 3 days post-transduction (FIG. 5a).

[0111] Flow cytometry analysis. Cells were processed 3 days post-transduction and stained with the cell viability dye 7-AAD for 5 min prior to flow cytometry analysis. Fluorescent light was measured by a BD LSRFortessa using a blue laser with bandpass filters 695 nm/40 mW and 530 nm/30 mW to detect the fluorochromes of 7-AAD and GFP, respectively (FIG. 5a).

[0112] Results & Discussion:

[0113] The results presented in FIGS. 5a and 5b demonstrate that increasing concentrations of CQ increase the number of GFP expressing cells up to 12 uM. The results also demonstrate that CQ increases the number of GFP expressing cells in a dose dependent manner.

[0114] The results presented demonstrate that the antimalarial agent, CQ, improves the efficacy of AAV transduction.