Genetic Construct

Abstract

The invention relates to the use of genetic constructs, expression cassettes and recombinant vectors comprising such constructs and cassettes for gene therapy and methods for treating neurodegenerative disorders, such as Parkinson's disease (PD). The constructs comprise 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). 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). The construct is delivered to the cerebrospinal fluid (CSF) of the subject.

Claims

1-26. (canceled)

27. A method of treating, preventing, or ameliorating a neurodegenerative disorder in a subject, wherein the method comprises administering, to a subject in need of such treatment, 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), and wherein the construct is delivered to the cerebrospinal fluid (CSF) of the subject.

28. The method according to claim 27, wherein the construct is delivered to the CSF by injection.

29. The method according to claim 27, wherein the genetic construct is delivered to the CSF via one or more of a group selected from: the intracerebral ventricle system; the cisterna magna; and between lumbar vertebrae L3/L4, L4/L5 or L5/S1.

30. The method according to claim 27, wherein the genetic construct is delivered to the CSF via the intracerebral ventricle system.

31. The method according to claim 27, wherein the genetic construct is delivered to the CSF via the cisterna magna.

32. The method according to claim 27, wherein the genetic construct is delivered to the CSF via between lumbar vertebrae L3/L4, L4/L5 or L5/S1.

33. The method according to claim 27, wherein the CSF DOPA level is increased sufficiently to trigger feedback inhibition of dopamine production by surviving dopaminergic cells within the striatum.

34. The method according to claim 27, wherein the CSF DOPA level is increased to between 5 pmol/ml and 20 pmol/ml, between 7 pmol/ml and 15 pmol/ml, or between 8 pmol/ml and 12 pmol/ml.

35. The method according to claim 27, wherein the genetic construct is delivered to the CSF by injection between lumbar vertebrae L3/L4, L4/L5 or L5/S1, wherein the method further comprises injecting a contrast media in combination with the genetic construct.

36. The method according to claim 27, wherein the neurodegenerative disorder to be treated is a disease associated with catecholamine dysfunction.

37. The method according to claim 27, wherein the neurodegenerative disorder to be treated is selected from the group consisting of Parkinson's disease, DOPA responsive dystonia, vascular Parkinsonism, side effects associated with L-DOPA treatment, or L-DOPA induced dyskinesia.

38. The method according to claim 27, wherein the neurodegenerative disorder to be treated is Parkinson's disease.

39. The method according to claim 27, 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.

40. The method according to claim 27, 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.

41. The method according to claim 27, wherein the construct further comprises a third coding sequence, which encodes 6-pyruvoyltetrahydropterin (PTPS), wherein the third coding sequence is 3′ to the second coding sequence and is part of the a single operon.

42. The method according to claim 41, wherein the third coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO: 32, 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: 33, or a fragment or variant thereof.

43. The method according to claim 27, 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.

44. A method of treating, preventing, or ameliorating a neurodegenerative disorder in a subject, wherein the method comprises administering, to a subject in need of such treatment, a recombinant vector comprising the genetic construct according to claim 27, wherein the vector is delivered to the cerebrospinal fluid (CSF) of the subject.

45. The method according to claim 44, wherein the recombinant vector is a recombinant AAV vector, or wherein the vector does not comprise a modified capsid.

46. A method of treating, preventing, or ameliorating a neurodegenerative disorder in a subject, wherein the method comprises administering, to a subject in need of such treatment, a pharmaceutical composition comprising the genetic construct defined in claim 27 and a pharmaceutically acceptable vehicle, wherein the pharmaceutical composition is delivered to the cerebrospinal fluid (CSF) of a subject.

47. A method of treating, preventing, or ameliorating a neurodegenerative disorder in a subject, wherein the method comprises administering, to a subject in need of such treatment, a pharmaceutical composition comprising the recombinant vector defined in claim 44 and a pharmaceutically acceptable vehicle, wherein the pharmaceutical composition is delivered to the cerebrospinal fluid (CSF) of a subject.

Description

[0184] FIG. 1 is a plasmid map of a first embodiment of the construct of the invention, showing the features of SEQ ID NO: 13;

[0185] FIG. 2 is a plasmid map of a second embodiment of the construct of the invention, showing the features of SEQ ID NO: 14;

[0186] FIG. 3 is a plasmid map of a third embodiment of the construct of the invention, showing the features of SEQ ID NO: 15;

[0187] FIG. 4 is a plasmid map of a fourth embodiment of the construct of the invention, showing the features of SEQ ID NO: 16;

[0188] FIG. 5 is a plasmid map of a fifth embodiment of the construct of the invention, showing the features of SEQ ID NO: 17;

[0189] FIG. 6 is a plasmid map of a sixth embodiment of the construct of the invention, showing the features of SEQ ID NO: 18;

[0190] FIG. 7 is a plasmid map of a seventh embodiment of the construct of the invention, showing the features of SEQ ID NO: 19;

[0191] FIG. 8 is a plasmid map of an eighth embodiment of the construct of the invention, showing the features of SEQ ID NO: 20;

[0192] FIG. 9 shows that rats treated by intrathecal injection of one embodiment of the construct of the invention display increased L-DOPA levels in the CSF after 14 days. Pre-AAV baseline DOPA is the first control referring to the DOPA concentration at the initial time-point of injection (pre-AAV), and the second control is the level of DOPA in a subject not exposed to the construct at 14 days (no AAV);

[0193] FIG. 10 shows that rats treated by intrathecal injection of one embodiment of the construct of the invention display increased dopamine levels in the CSF. The control refers to the dopamine concentration at the initial time-point of injection (pre-AAV); and

[0194] FIG. 11 shows that rats treated by intrathecal injection of one embodiment of the construct of the invention display reduced intracellular dopamine levels in the striatum. The control refers to the dopamine concentration 14 days after AAV administration (14 day no AAV control).

[0195] FIG. 12 shows that intrathecal injection into either the lateral intracerebral ventricle or the cisterna magna produced a similar reduction in striatal intracellular dopamine levels.

EXAMPLES

Background

[0196] Previous studies for gene therapy for Parkinson's disease have assumed that for successful treatment, vectors for gene therapy would need to be transferred directly into the patient's striatum, where the vector carries genes necessary for the production of dopamine or L-DOPA by brain cells that would ordinarily be non-dopamine producing. The aim of such treatment is the local generation of dopamine within the affected areas of the brains of Parkinson's patients. Several methods of gene therapy have been disclosed. However, while the technique has shown some promise, and the previous methods provide a proof of the principle, previous vectors have not been optimal, and are associated with brain surgery risks. In particular, there has been a need for vectors and delivery means that leads to optimal production of dopamine (either directly or indirectly via L-DOPA) in the brains of Parkinson's patients, and which can be manufactured at suitable levels and with suitable cost effectiveness to be a viable treatment option, and which do not suffer the risks and complexities associated with direct injection into the striatum, putamen, caudate or substantia nigra.

[0197] The inventor hypothesised that by injecting the AAV into the intrathecal space—i.e. into the cerebrospinal fluid—it is possible to raise the CSF and brain extracellular fluid levels of L-DOPA and use this as a route of impacting the dopamine level in the striatum of patients with PD. Although this would expose the entire brain to increased levels of L-DOPA, this should be similar to what happens when patients are treated with classical oral L-DOPA. The latter has been the gold standard for the treatment of PD for more than 40 years and the “whole brain” impact of L-DOPA is usually well-tolerated in the majority of patients.

[0198] Based on the inventor's hypothesis, he performed a study in rats using two routes to administer constructs of the invention into the CSF, either a single simple injection into the intracerebral ventricle system or a single simple injection into the cisterna magna.

[0199] Materials and Methods

[0200] Construct/Vector

[0201] A bicistronic AAV (serotype 9) was used prepared by triple transfection. The vector genome included a CMV enhancer, CMV promoter, cDNA for human tyrosine hydroxylase (excluding the regulatory domain), an F2A linker, furin cleavage site, cDNA for human GCH1, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) modified to prevent expression of X-protein and SV40pA, in series between two AAV2 inverted terminal repeats (ITRs).

[0202] OHDA Lesion of the MFB

[0203] Unilateral lesions of the nigrastriatal pathway were conducted by intracerebral administration of 6-hydroxydopamine (6-OHDA). 6-OHDA was formulated at 5 mg/ml solution in 0.03% ascorbic acid in sterile 0.90% NaCl. Three μL of 6-OHDA was injected into the medial forebrain bundle at the following stereotaxic coordinates from bregma: Anteroposterior (A/P) −4.0 mm; mediolateral (M/L) −1.3 mm; ventrodorsal (V/D) −8.0 mm with reference to top of skull.

[0204] ICV Injection of TA and CSF Collection

[0205] Two weeks after 6-OHDA lesion, animals were randomized into the treatment groups. Animals from Group 2 were anesthetized with isoflurane and placed in the stereotaxic frame with a nose bar set a +5 mm. A 2 cm sagittal incision was made to locate bregma. A hole was drilled using the following coordinates: AP: −0.4; L: +2.0. CSF (˜50 μl) was drawn from the ventricle (using a Hamilton syringe lowered at −4.5 mm).

[0206] For CM collection, rats were anesthetized with isoflurane and positioned in the stereotaxic frame. The rat head was flexed downward at approximately 45 degrees, a depressible surface with the appearance of a rhomb between occipital protuberances and the spine of the atlas was visible. The 23 G needle was punctured into the cisterna magna for CSF collection without making any incision at this region.

[0207] The AAV9 vector was slowly infused into the ventricle (10 μl/min) using the same coordinates and the same hole, the volume of injection: 50 μl (TBD). The needle was be left in place for 3 min and then withdrawn. The incision was closed with wound clips. After CM CSF collection, the needle was left in place and then connected to a syringe containing the TA. TA was slowly infused into the CM (10 μl/min) the volume of injection: ˜50 μl (TBD). The needle was left in place for 3 min and then withdrawn. Control animals did not have vector injected.

[0208] Terminal CSF Collection and Striatum Dissection

[0209] On day 28 days after the 6-OHDA lesions, animals were anesthetized with isoflurane and CSF was collected from the CM, transferred into a clean tube and flash frozen. After CSF collection, animals were sacrificed and brains extracted. Left striatum was dissected, weighed in the tube and flash frozen CSF samples were stored at −80° C. until shipment to client-designated laboratory.

[0210] Table 1 shows a summary of the steps that were performed to measure CSF levels after lesion of the basal forebrain and subsequent injection of the bicistronic vector.

TABLE-US-00045 Day Event Day 1  Surgery/unilateral lesion of the medial forebrain bundle Day 14 Surgery: CSF collection and TA injection into the lateral ventricle or CM Day 28 Takedown: CSF collection, striatum dissection

[0211] Results and Discussion

[0212] FIGS. 1 to 8 show embodiments of the gene therapy vectors used in accordance with the invention described herein. In particular, the vector shown in FIG. 3 was used in the following examples.

Example 1—DOPA Levels are Elevated in the CSF

[0213] The gene therapy vectors described herein are designed to transfect cells of the ependyma and the adjacent tissue in the vicinity of the CSF. The vectors transduce production of tyrosine hydroxylase and GCH1 (the latter is rate-limiting in the production of BH4, which is a cofactor essential for TH activity). FIG. 9 shows that DOPA (=L-DOPA) levels in the CSF display a very highly significant increase in vector-treated animals compared with either pre-treatment or non-treated (no vector) controls.

Example 2—Dopamine Levels are Elevated in the CSF

[0214] It is known that there is residual AADC activity in the Parkinsonian brain, and the fact that oral L-DOPA is active depends on this. While there are a number of views on where this AADC resides (e.g. surviving dopaminergic neurons, interneurons, serotonegic neurons, or a combination of these), the inventor has observed the increased CSF L-DOPA to result in an increase in CSF dopamine concentrations due to this decarboxylation. Indeed, FIG. 10 shows that Dopamine levels in the CSF display a very highly significant increase in vector-treated animals compared with the pre-treatment control.

Example 3—Striatum Intracellular Dopamine Levels are Reduced

[0215] The DOPA and Dopamine produced in this way in the CSF, ependyma and adjacent tissue will be distributed more broadly into the brain via blood or in extracellular fluid pulsating in the perivascular space, and this will enable them to reach the striatum to impart their therapeutic effects. The striatum can be viewed as two compartments (the intracellular compartment and the extracellular fluid compartment), and it will be appreciated that what happens in the extracellular compartment influences what happens intracellularly. In the present invention, dopaminergic cells can detect the amount of dopamine in the extracellular fluid within the striatum. If the extracellular level of dopamine is high, the striatal cells react by reducing their production and subsequent secretion of dopamine.

[0216] Assaying the intracellular Dopamine levels in the striatum therefore provides an indicator of whether the increase in L-DOPA production in the ependyma and tissue adjacent to the CSF is: [0217] (a) distributed to non-adjacent tissue; and [0218] (b) sufficient to stimulate dopamine receptors at these non-adjacent sites and therefore to be of therapeutic potential.

[0219] FIG. 11 shows that the intracellular striatum Dopamine levels display a very highly significant decrease in vector treated animals compared with the no AAV control. As the intracellular levels of Dopamine are reduced in the vector-treated animals, this is consistent with a subsequent increase in extracellular Dopamine levels. Given that Figure C shows the concentration of intracellular striatal Dopamine concentrations, and the understanding that the therapeutic objective with this invention is to raise L-DOPA levels in the extracellular fluid surrounding the basal ganglia (including the striatum), these data clearly support the view that the vector is achieving its desired effect in that the increases in DOPA and Dopamine are principally in the extracellular fluid compartment of the striatum. The increase in DOPA and Dopamine in the extracellular compartment will result in feedback inhibition of Dopamine production within the surviving dopaminergic cells of the lesioned striatum.

[0220] FIG. 12 shows that intrathecal injection into either the lateral intracerebral ventricle or the cisterna magna produced a similar reduction in striatal intracellular dopamine levels.

SUMMARY

[0221] In summary, the use of the constructs described herein displays the following advantages over current methods in the art:

[0222] i) the invention is a simple and practical method of treating Parkinson's which addresses the limitations of previously employed methods. The inventor has demonstrated that a gene therapy construct administered in non-targeted manner into the CSF can result in an increase is substrate (DOPA) sufficient to enable local conversion of the neurotransmitter L-DOPA within the therapeutic target (the striatum) and has demonstrated that the resulting extracellular levels of dopamine are sufficient to stimulate and expected result on local dopamine receptors. (ii) provision of constant level of L-DOPA substrate to the CNS. This may replace or reduce the need for oral L-DOPA therapy. By providing a constant level of L-DOPA production, the peaks and troughs associated with oral therapy will be avoided or reduced. This in turn will prevent, or reduce the risk of, or treat long-term complications of L-DOPA therapy that are related to the variable blood levels associated with oral L-DOPA therapy (including dyskinesia, on/off fluctuations and “freezing”);

[0223] iii) no need for the requirement of complex, lengthy surgery to infuse gene therapy directly to the striatum. Current gene therapy approaches seeking to increase L-DOPA or dopamine production within the central nervous system infuse vector directly into the striatum. This may require use of multiple needle tracts though brain tissue of both hemispheres in order to ensure adequate distribution of vector over the target tissue. Infusion of vector into brain tissue must be slow to achieve maximum distribution and avoid injury. The resulting procedure must be implemented by a full neurosurgical team in a neurosurgical suite and may take up to 10 hours (usually 4-6 hours). The procedure carries the risk of death or incapacity due to cerebral haemorrhage. In contrast direct injection of vector into the cerebrospinal fluid can be achieved more quickly and simply and at lower risk;

[0224] iv) marked reduction in cost of goods versus gene therapy transducing constant peripheral production of L-DOPA (for example from liver and/or muscle). By enabling local production of L-DOPA within the CNS, the invention avoids inefficiency due to peripheral distribution, excretion and metabolism of L-DOPA before it reaches the CNS and reduces the challenge of transfer of L-DOPA across the blood brain barrier. The invention therefore requires a lower dose of vector with a lower cost of goods. The invention avoids the need for many intramuscular injections or complex infusion regimens necessary to adequately transduce liver or muscle and may be less immunogenic;

[0225] v) the use results in the production of L-DOPA but does not transduce expression of AADC. Thus, while increasing the level of the dopamine substrate available throughout the CNS (as happens with current standard therapy with oral or enteral administration of L-DOPA) production of dopamine is only increased in areas of brain with significant intrinsic AADC activity. This reduces the risk of off-target dopamine induced toxicity; and

[0226] vi) by providing constant levels of DOPA and dopamine in the striatal extracellular fluid the invention achieves the same pharmacological objective as currently achieved by continuous infusion of L-DOPA/carbidopa gel without the need for continuous infusion into the jejunum. The invention will enable the superior efficacy achieved by continuous infusion of L-DOPA/carbidopa gel (Duodopa) but without the lifelong burden of PEG tube and the associated risks of blockage, displacement and infection.