Genetic Construct

Abstract

A genetic construct comprises a promoter operably linked to a first coding sequence, which encodes tyrosine hydroxylase (TH), and a second coding sequence, which encodes GTP cyclohydrolase 1 (GCH1), wherein the second coding sequence is 3 to the first coding sequence, and the first and second coding sequences are part of a single operon. The genetic construct does not encode aromatic amino acid decarboxylase (AADC).

Claims

1. A genetic construct comprising a promoter operably linked to a first coding sequence, which encodes tyrosine hydroxylase (TH), and a second coding sequence, which encodes GTP cyclohydrolase 1 (GCH1), wherein the second coding sequence is 3 to the first coding sequence, and the first and second coding sequences are part of a single operon, wherein the genetic construct does not encode aromatic amino acid decarboxylase (AADC).

2. A genetic construct according to claim 1, wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO: 1 or SEQ ID No:2, or a fragment or variant thereof, and/or comprises a nucleotide sequence encoding an amino acid sequence substantially as set out in SEQ ID NO: 21 or SEQ ID No:22, or a fragment or variant thereof.

3. A genetic construct according to claim 1, wherein the second coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO: 4, or a fragment or variant thereof, and/or comprises a nucleotide sequence encoding an amino acid sequence substantially as set out in SEQ ID NO: 23, or a fragment or variant thereof.

4. A genetic construct according to claim 1, wherein the promoter is a constitutive promoter, an activatable promoter, an inducible promoter, or a tissue-specific promoter, optionally wherein the promoter comprises a CMV promoter or a human synapsin promoter.

5. A genetic construct according to claim 1, wherein the promoter comprises a nucleotide sequence substantially as set out in SEQ ID No: 5 or 25, or a fragment or variant thereof.

6. (canceled)

7. A genetic construct according to claim 1, wherein the genetic construct comprises a spacer sequence disposed between the first and second coding sequences, and the spacer sequence is such that it allows the production of functional TH and the production of functional GCH1 from the single promoter.

8. A genetic construct according to claim 7, wherein the spacer sequence comprises an IRES, optionally wherein the IRES comprises a nucleotide sequence substantially as set out as SEQ ID NO: 6 or 7 or a fragment or variant thereof.

9. A genetic construct according to claim 7, wherein the spacer sequence comprises a viral 2A peptide spacer.

10. A genetic construct according to claim 9, wherein the spacer sequence comprises a furin cleavage site, optionally wherein the spacer sequence comprises a nucleotide sequence substantially as set out in SEQ ID No:8, or a fragment or variant thereof.

11. A genetic construct according to claim 7, wherein the spacer sequence comprises a flexible linker, which allows for the expression of both TH and GCH1 as a single polypeptide chain, wherein the TH and GCH1 act as independent proteins, optionally wherein the flexible linker sequence comprises a nucleotide sequence substantially as set out in SEQ ID No: 9, or a fragment or variant thereof.

12. A genetic construct according to claim 1, wherein the genetic construct comprises: (i) a nucleotide sequence encoding Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE), optionally wherein the WPRE comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 10 or SEQ ID NO: 11, or a fragment or variant thereof; (ii) a nucleotide sequence encoding a polyA tail, optionally wherein the polyA tail comprises a nucleic acid sequence substantially as set out in SEQ ID No: 12, or a fragment or variant thereof; and/or (iii) left and/or right Inverted Terminal Repeat sequences (ITRs).

13.-14. (canceled)

15. A genetic construct according to claim 1, wherein the construct comprises a sequence substantially as set out in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, or a fragment or variant thereof.

16. A recombinant vector comprising the genetic construct according to claim 1.

17. The recombinant vector according to claim 16, wherein the recombinant vector is a recombinant AAV vector.

18. The recombinant vector according to claim 16, wherein the recombinant vector comprises a sequence substantially as set out in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or a fragment or variant thereof.

19. A method of treating, preventing, or ameliorating a neurodegenerative disorder in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of the genetic construct according to claim 1.

20.-21. (canceled)

22. The method of claim 19, wherein the neurodegenerative disorder is a disease associated with catecholamine dysfunction, optionally wherein the disease associated with catecholamine dysfunction is characterised by a dopamine deficiency.

23. (canceled)

24. The method of claim 22, wherein the neurodegenerative disorder is Parkinson's disease.

25. A pharmaceutical composition comprising the genetic construct according to claim 1, and a pharmaceutically acceptable vehicle.

26. (canceled)

Description

[0146] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figure, in which:

[0147] FIG. 1 is a graphical representation of the results of an in vitro assay comparing the TH expression between various embodiments of the constructs of the invention and those described in prior art references;

[0148] FIG. 2 is a graphical representation of the results of an in vitro assay comparing the GCH1 expression between various embodiments of the construct of the invention and those described in prior art references;

[0149] FIG. 3 is a plasmid map of a first embodiment of the construct of the invention, showing the features of SEQ ID NO: 13;

[0150] FIG. 4 is a plasmid map of a second embodiment of the construct of the invention, showing the features of SEQ ID NO: 14;

[0151] FIG. 5 is a plasmid map of a third embodiment of the construct of the invention, showing the features of SEQ ID NO: 15;

[0152] FIG. 6 is a plasmid map of a fourth embodiment of the construct of the invention, showing the features of SEQ ID NO: 16;

[0153] FIG. 7 is a plasmid map of a fifth embodiment of the construct of the invention, showing the features of SEQ ID NO: 17;

[0154] FIG. 8 is a plasmid map of a construct corresponding to the construct of WO2011/054976, and used herein as a reference plasmid;

[0155] FIG. 9 is a graphical representation of the results of an in vitro assay comparing TH expression at various amounts of DNA;

[0156] FIG. 10 is a plasmid map of a sixth embodiment of the construct of the invention, showing the features of SEQ ID NO: 18;

[0157] FIG. 11 is a plasmid map of a seventh embodiment of the construct of the invention, showing the features of SEQ ID NO: 19;

[0158] FIG. 12 is a plasmid map of an eighth embodiment of the construct of the invention, showing the features of SEQ ID NO: 20;

[0159] FIG. 13A is a plasmid map of a monocistronic construct which includes TH;

[0160] FIG. 13B is a plasmid map of a monocistronic construct which includes GCH1;

[0161] FIG. 14 is a graphical representation of the in vivo stepping assay control experiment showing baseline stepping in rats post-lesion; and

[0162] FIG. 15 is a graphical representation of the in vivo stepping assay showing a step count improvement in rats treated with a construct of the invention, when compared to a prior art construct, 4 weeks post treatment.

EXAMPLES

[0163] The inventor has investigated gene therapy for treating neurodegenerative disorders, including dopamine deficiency disorders, and Parkinson's disease. The inventor has developed a novel genetic construct and associated AAV vector for use in treating Parkinson's disease.

Example 1Assessment of Tyrosine Hydroxylase Gene Expression

Background

[0164] A reference plasmid corresponding to the prior art plasmid of WO 2011/054976 A2 was utilised in this study. This plasmid is graphically depicted in the map of FIG. 8.

[0165] The reference plasmid contains two separate expression cassettes for the murine GTH cyclohydrase 1 (GCH1) and human tyrosine hydroxylase (TH) genes. The purpose of the study was to compare TH expression of the aforementioned reference plasmid at the mRNA level versus three embodiments of the present invention, or a combination of two plasmids, each expressing a single gene, so as to identify a dual gene expression plasmid with improved TH expression.

Preliminary Experiment

[0166] HEK 293 cells were transfected with five different amounts of the reference plasmid, followed by RNA extraction, cDNA synthesis, and subsequent assessment of mRNA expression of GAPDH (housekeeping gene) and human TH by qPCR. The results of this assay are shown in FIG. 9.

[0167] Three DNA amounts, 0.25 g, 0.125 g and 0.0625 g, were selected for use in further experiments.

[0168] Comparison between the prior art constructs and embodiments of the invention HEK 293 cells were transfected, at the three DNA doses selected from the preliminary experiment, with either the reference plasmid or the four test conditions (Test1-Test4). This was followed by RNA extractions, cDNA synthesis and subsequent assessment of mRNA expression of GAPDH (housekeeping gene) and human tyrosine kinase by qPCR. The sample Ref 0.0625 g was selected as the calibrator sample for qPCR analysis.

[0169] Test1 comprised transfection with a plasmid of a sequence according to SEQ ID NO: 18. This plasmid is graphically depicted in the map of FIG. 10. This construct comprises a sequence encoding TH, an IRES, and a downstream sequence encoding GCH1.

[0170] Test2 comprised transfection with a plasmid of a sequence according to SEQ ID NO: 19. This plasmid is graphically depicted in the map of FIG. 11. This construct comprises a sequence encoding TH, a furin cleavage site, a viral 2A peptide spacer, and a downstream sequence encoding GCH1.

[0171] Test3 comprised transfection with a plasmid of a sequence according to SEQ ID NO: 20. This plasmid is graphically depicted in the map of FIG. 12. This construct comprises a sequence encoding TH, a flexible linker, and a downstream sequence encoding GCH1.

[0172] Test4 comprised transfection involving co-administration of a plasmid graphically depicted in the map of FIG. 13A and of a plasmid graphically depicted in the map of FIG. 13B. One construct encodes TH (FIG. 13A), whereas the other construct encodes GCH1 (FIG. 13B). Test4 was to allow the testing of the expression achieved by the above bicistronic constructs over these two co-administered monocistronic constructs.

[0173] The results of this experiment are shown in FIG. 1 and Table 1. Test1, Test2, and Test3 (embodiments of the present invention) were all improved over the reference plasmid. Test1 resulted in an up to 9.4-fold increase in mRNA expression over the reference plasmid. It is notable that if sample Ref 0.0625 g were used as the calibrator sample, up to 9.9-fold increase in mRNA level could be detected.

TABLE-US-00032 TABLE 1 Assessment of Tyrosine Hydroxylase gene expression Av SD Ref 0.25 g 1 0.032029 Ref 0.125 g 0.481388 0.024329 Ref 0.0625 g 0.261409 0.033758 Test1 0.25 g 9.411235 0.182147 Test1 0.125 g 4.417566 0.702172 Test1 0.0625 g 2.587243 0.722136 Test2 0.25 g 4.161938 2.724108 Test2 0.125 g 3.838677 2.227054 Test2 0.0625 g 1.415666 0.075599 Test3 0.25 g 2.553369 0.039338 Test3 0.125 g 1.326091 0.157098 Test3 0.0625 g 0.574204 0.077967 Test4 0.25 g 2.877203 0.380339 Test4 0.125 g 1.401549 0.002572 Test4 0.0625 g 1.009721 0.216553

[0174] Surprisingly, Test1 and Test2 were also improved over Test4 (the co-administered monocistronic constructs). This is particularly advantageous as therapy with a single biscistronic construct has further advantages over therapy using two monocistronic constructs. These advantages are as disclosed herein, but in short the bicistronic construct ensures that the genes are delivered to the same cell and also has advantages in manufacturing and economy.

Example 2Assessment of Murine GTP Cyclohydrolase 1 Gene Expression

[0175] The purpose of this study was to quantify expression of the murine GCH1 gene from vectors featuring a number of gene configurations. In particular, two embodiments of the invention were compared to a reference plasmid corresponding to the prior art plasmid of WO2011/054976 A2.

[0176] The reference plasmid was the same as that described in Example 1 and FIG. 8.

[0177] The plasmid used in Test1 was the same as that described in Example 1, FIG. 10, and SEQ ID NO: 18.

[0178] The plasmid used in Test2 was the same as that described in Example 1, FIG. 11, and SEQ ID NO: 19.

[0179] The results of this experiment are shown in FIG. 2 and Table 2. Test1 and Test2 (embodiments of the present invention) were improved over the reference plasmid. Notably, an up to 2.9-fold increase in mRNA expression was observed for Test1 plasmid over the reference plasmid, and if using sample Ref 0.0625 g as the calibrator sample, up to 3.3-fold increase in mRNA level could be detected.

TABLE-US-00033 TABLE 2 Assessment of murine GTP cyclohydrolase 1 gene expression Av SD Ref 0.25 g 1 0.016089 Ref 0.125 g 0.452139 0.040327 Ref 0.0625 g 0.244783 0.037694 Test1 0.25 g 2.874335 0.029164 Test1 0.125 g 1.309529 0.077492 Test1 0.0625 g 0.8055 0.187179 Test2 0.25 g 1.289959 0.887127 Test2 0.125 g 1.226948 0.708271 Test2 0.0625 g 0.442867 0.021152

Example 3In Vivo Assessment of the Construct of the Invention

[0180] The purpose of this study was to compare the ability of the inventor's construct (MRX001, FIG. 3, SEQ ID NO: 13) expressing TH and GCH1 by the same promoter with a comparator based on the prior art construct expressing the two genes by separate promoters, to improve the step count in a rat model of Parkinson's disease, using the gold standard technique of the art.

Materials and Methods

[0181] The methods used in this example are described in detail in Cederfjall et al, Scientific Reports, 2013; 3: 2157. Therefore, this experiment represents a robust comparison with the closest prior art construct.

[0182] Female Sprague Dawley rats, weighing 200-250 g were used. All rats received unilateral 6-OHDA medial forebrain bundle lesions. Rats were tested for 3 and 4 week post-lesion amphetamine rotations to confirm adequate lesioning. Rats were allocated to the treatment groups to ensure that the magnitude of lesion effect was evenly distributed between the two groups. Rats received intrastriatal infusions of the reference vector (Promoter-GCH1-Promoter-TH) (n=8), the experimental vector (MRX) (n=8) or remained as lesion only controls (n=.sub.4).

[0183] Each animal in each vector group received a total of 2109 genome copies of vector in 5 ml of vehicle This was intended to be not to fully restore motor function i.e to be sufficiently high to show activity but not sufficient to achieve optimal results and leaves a clear window to demonstrate enhanced efficacy if present in the comparator. The injections were made at two sites with two 1.5 ml deposits in the ventral tract and two 1.0 ml deposits in the dorsal injection tract. The coordinates were: (1) AP: 11.0 mm; ML: 2.8 mm and DV: 4.5, 3.5 mm and (2) AP: 0.0 mm; ML: 4.0 mm and DV: 5.0, 4.0 mm with the tooth bar set to 2.4 mm. The AAV vector was injected at a speed of 0.4 ml/min and the needle was kept in place for 1 min after the ventral and 3 min after the dorsal deposit was delivered, before it was slowly retracted.

[0184] Stepping tests were performed according to the method described in Olsson et al. (Olsson, M., et al. Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test. J Neurosci 15, 3863-3875 (1995). An investigator blinded to the group identities of the animals assessed forelimb use by holding the rat with two hands only allowing one forepaw to reach the table surface. The animal was then moved sideways over a defined distance at a constant speed over 4-5 sec. The investigator scored the numbers of adjusting side steps in the forehand direction twice on a testing day, and the average was calculated. The primary endpoint was prospectively defined as the difference in step count at week 4 post injection of vector.

Results

[0185] The results of this experiment are shown in FIGS. 14 and 15. Surprisingly, the inventor has found that the claimed construct is capable of partially restoring the step count in mice models of Parkinson's disease, whereas the prior art construct provides no such effect. This result demonstrates that the inventor's construct is significantly superior, and represents an important improvement in the art.

CONCLUSIONS

[0186] WO2013/061076 and WO2010/055209 (Oxford Biomedica) identified three proteins that were potentially effective in the treatment of Parkinson's disease, and all three proteins were regarded as being essential to provide a therapeutic effect. There is no discussion on the advantages of using a single promoter approach, as claimed, over a separate promoter approach. In fact, on page 3 in WO2013/06 1076, in the last line of the second paragraph, it is stated that: . . . increased levels of L-DOPA and dopamine were not due to increases in protein expression from the fusion design vectors . . . . Thus, these documents teach that, TH, GCH1 and AADC are required to produce a therapeutic effect. It is silent on the advantages or disadvantages of different promoter set-ups, as are all other relevant publications in relation to such constructs.

[0187] Cederfjall and Kirik (including WO2015/152813) moved the art forward to some extent by identifying that only two proteins were required to produce a therapeutic effect, these being TH and GCH1. However, the expression of these two genes is driven by separate promoters, and while WO2015/152813 suggests that this may be an effective means of expressing these genes for therapy, subsequently published work has proven that this approach is ineffective. The separate promoter approach surprisingly results in is sub-optimal expression when translated to results outside of that of murine studies. So much so that it failed to achieve complete reversal of motor symptoms in a primate model of Parkinson's disease. As a result of this defect, the corresponding product never went into full development, and Cederfjall et al. (Scientific reports; 3: 2157; 8 Jul. 2013) stated that this problem requires a solution prior to the initiation of clinical trials utilizing this approach.

[0188] Surprisingly, and without wishing to be bound to any particular theory, the inventor has discovered that a separate promoter approach, as used in the prior art, results in random interference between the promoters, such that the expression of one or both genes is reduced to sub-therapeutic levels. This is not described in the any prior art document in the field, and would not have been considered by the skilled person looking to solve the problem of providing an improved TH and GCH1 expression construct.

[0189] The inventor has developed a novel construct for expression of TH and GCH1 (and not AADC), wherein expression of the two genes is driven by a single promoter. This results in significantly higher levels of expression than that of the construct of WO2015/152813. For example, the in vitro data provided in FIGS. 1 and 2 compare the expression levels of TH and GCH1 with that of the control construct which places the same promoter before each gene in the same manner as described WO2015/152813. This comparative data shows that the claimed invention displays surprisingly higher levels of expression of both TH and GCH1.

[0190] The in vivo experiments performed in a murine model (FIGS. 14 and 15) confirm the in vitro findings. The methods used in the inventor's in vivo experiments are the same as those disclosed in Cederfjall et al, 2013, the authors being the inventors of WO2015/152813. Therefore, this experiment represents a robust comparison with the closest prior art construct. The control construct places the same promoter before each gene in the same manner as described, in WO2015/152813. The inventor performed the experiments at titers that are sufficiently high to show activity, but not sufficient to achieve optimal results, thus leaving a clear window to demonstrate the enhanced efficacy.

[0191] Surprisingly, the inventor has found that the claimed construct is capable of restoring the step count in mice models of Parkinson's disease, whereas construct incorporating a promoter before each gene shows no effect. Moreover, it is particularly surprising that this bicistronic construct has been demonstrated by the inventor to be markedly more effective in vitro than a 1:1 mixture of equivalent tires of a monocistronic TH vector and a monocistronic GCH1 vector.

[0192] This result demonstrates that the inventor's construct is far superior, and represents a significant improvement in the art. The inventor's construct results in improved expression of TH and GH1 and this translates to an improved therapeutic effect, as evidenced by the in vitro and in vivo data provided herein.