U.S. patent application number 16/952364 was filed with the patent office on 2021-07-15 for gene therapies for neurodegenerative disorders targeting ganglioside biosynthetic pathways.
The applicant listed for this patent is THOMAS JEFFERSON UNIVERSITY. Invention is credited to David W. Anderson, Jay S. Schneider.
Application Number | 20210213142 16/952364 |
Document ID | / |
Family ID | 1000005489867 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210213142 |
Kind Code |
A1 |
Schneider; Jay S. ; et
al. |
July 15, 2021 |
GENE THERAPIES FOR NEURODEGENERATIVE DISORDERS TARGETING
GANGLIOSIDE BIOSYNTHETIC PATHWAYS
Abstract
A composition of matter comprising an adeno-associated virus
(AAV) or other human compatible virus, encoding the gene for
Sialidase Neu3, B3Galt4, St3Gal2, or combinations thereof, and a
neuron specific promoter, wherein the composition is suitable for
administration to a patient comprising injecting the AAV or other
human compatible virus into the brain by intracranial stereotaxic
injunction; wherein the AAV's encoding for the Sialidase Neu3,
B3Galt4, St3Gal2, or combinations thereof enhance and/or normalize
levels of GM1 in neurons, providing both therapeutic relief and
disease modifying effects in specific areas of the brain relevant
to particular neurodegenerative diseases.
Inventors: |
Schneider; Jay S.; (Cherry
Hill, NJ) ; Anderson; David W.; (Newtown Square,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THOMAS JEFFERSON UNIVERSITY |
Philadelphia |
PA |
US |
|
|
Family ID: |
1000005489867 |
Appl. No.: |
16/952364 |
Filed: |
November 19, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15746355 |
Jan 19, 2018 |
10874749 |
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PCT/US16/43393 |
Jul 21, 2016 |
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16952364 |
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62194954 |
Jul 21, 2015 |
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62194910 |
Jul 21, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/531 20130101;
A61K 31/7088 20130101; C12N 2015/8518 20130101; C12Q 1/6883
20130101; A61K 45/06 20130101; A61K 48/0058 20130101; C12N 2320/31
20130101; C12N 2750/14143 20130101; C12N 15/85 20130101; A61K 35/30
20130101; C12Q 1/68 20130101; A61K 48/0033 20130101; C12Y 204/99004
20130101; C12Q 2600/158 20130101; A01K 2267/0318 20130101; A01K
2207/20 20130101; A61K 48/005 20130101; A61K 48/0075 20130101; A01K
2227/105 20130101; C12N 15/1137 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 35/30 20060101 A61K035/30; C12N 15/113 20060101
C12N015/113; C12Q 1/6883 20060101 C12Q001/6883; A61K 45/06 20060101
A61K045/06; C12Q 1/68 20060101 C12Q001/68; A61K 31/7088 20060101
A61K031/7088; C12N 15/85 20060101 C12N015/85 |
Claims
1.-33. (canceled)
34. A method of treating neurodegenerative disease in a subject in
need thereof, the method comprising administering to said subject
an effective amount of a vector encoding sialidase Neu3; wherein
said vector modifies cells in said subject to increase production
of Neu3, thereby increasing the amount of GM1 in the brain of said
subject.
35. The method of claim 34, further comprising administering to
said subject a vector encoding B3Galt4.
36. The method of claim 34, further comprising administering to
said subject a vector encoding B3Galt4, St3Gal2, or combinations
thereof.
37. The method of claim 36 wherein said vector encoding sialidase
Neu3 and/or said vector encoding B3Galt4, St3Gal2, or combinations
thereof is a viral vector selected from the group consisting of
retrovirus, lentivirus, adenovirus, and adeno-associated virus
(AAV) vector.
38. The method of claim 36 wherein said vector encoding sialidase
Neu3 and/or the vector encoding B3Galt4, St3Gal2, or combinations
thereof is a non-viral vector.
39. The method of claim 34, further comprising concomitantly
administering to said subject a dietary supplement with complex
milk lipids (CMLs) having a concentrated dietary source of GM3.
40. The method of claim 34, further comprising concomitantly
administering shRNA/siRNA St3gal2 to increase GM1 levels.
41. The method of claim 34, wherein the neurodegenerative disease
is Parkinson's disease or Huntington's disease.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/194,910, filed Jul. 21, 2015 and
62/194,954, filed Jul. 21, 2015, the disclosure contents of which
are hereby incorporated by reference in their entirety.
FIELD OF INVENTION
[0002] The present application is generally related to therapeutic
treatments for neurodegerative disorders including Parkinson's
disease (PD) and Huntington's disease (HD) comprising
administration of a Sialidase Neu3 through gene therapy, through
administration of B3Galt4 cDNA or a combination thereof, to a
patient suffering from a neurodegenerative disease.
BACKGROUND OF THE INVENTION
[0003] HD is a hereditary autosomal-dominant, progressive
neurodegenerative disorder, resulting from expansion of a
polygultamine (CAG) repeat in the gene coding for the Huntingtin
(Htt) protein, leading to formation of mutant Htt (mHtt), which
confers a toxic gain of function, with neurons of the striatum
being particularly vulnerable. Accumulation of mHtt drives neuronal
dysfunction via transcriptional dysregulation, modification of cell
signaling, abnormal axonal transport and synaptic activity leading
to clinical signs and symptoms including chorea, dystonia,
incoordination, cognitive decline and behavioral difficulties,
culminating with death. Latency from diagnosis to death is
typically approximately 20 years. Death is commonly due to
complications of falling and/or dysphagia and aspiration, although
it is estimated that >25% of patients attempt suicide at some
point after diagnosis.
[0004] While genetic testing is available for HD, less than 5% of
at-risk individuals elect to have predictive testing, with many
citing the lack of an effective treatment as the rational for this
decision. Currently the standard of care for HD is symptomatic,
utilizing modestly effective anti-choreic drugs such as
tetrabenazine. The use of symptomatic therapy does not alter the
course of the disease. Survival rates in treated v. untreated
patients are similar. As such, there is a significant unmet medical
need for an effective, disease modifying therapy for HD.
[0005] Recent research found that as well as the mHtt toxicity in
striatal (and cortical) neurons in HD, there is a significant
dysregulation of ganglioside biosynthesis in these cells in HD
models. Similar defects were found in fibroblasts from HD patients,
suggesting that dysregulation in ganglioside expression may be a
universal phenomenon in HD. Gangliosides, sialic acid containing
sphingolipids, are major components of membrane lipid rafts where
they act as modulators of cell signaling. Found throughout the
body, gangliosides are highly enriched in the central nervous
system (CNS), where they are critical to normal CNS development and
neuron survival. Maglione et al. described impaired ganglioside
metabolism in tissues from HD patients (fibroblasts and brain) and
in a transgenic model of HD in mice (YAC128). These findings have
been confirmed by others and extended to the R6/1 transgenic HD
mouse, further strengthening the correlation between ganglioside
dysregulation and HD. Primarily, the monosialoganglioside (GM1) is
significantly reduced in both HD and PDmodels, although downstream
polysialogangliosides such as GD1a, GD1b, GT1b are also partially
affected. The administration of bovine brain-derived GM1 by
infusion pump to YAC128 mice that were symptomatic for motor
impairment resulted in normalization of motor function within 2
weeks of the start of treatment and lasting until the last day of
infusion and resulted in phosphorylation of mHtt at serine residues
13 and 16 that attenuated mHtt toxicity, suggesting a disease
modifying effect of GM1 administration.
[0006] Parkinson's disease is another neurodegenerative disorder
that slowly progresses and is characterized by loss of
dopamine-producing neurons in the substantia nigra, loss of
forebrain dopamine, and a time-dependent worsening of clinical
symptoms. Although symptomatic improvement can be obtained with
available pharmacotherapies, functional ability deteriorates over
time with the progressive loss of nigrostriatal dopamine neurons.
Development of therapies that can slow the progression of the
disease would fill a major unmet medical need in PD.
[0007] It is estimated that PD is the second most common
neurodegenerative disease of mid to late life, with estimates of 1
of every 200 persons in the US aged 60-69 having PD. Additionally,
1 in 100 persons over 70 and 1 in every 35 persons over 80 suffer
from PD. Worldwide, numbers suggest that between 4 to 5 million
people suffer from PD and that number could double to between 8-10
million by 2030.
[0008] Current gold-standard treatment for PD is a drug first
discovered in the 1960's, levodopa, which strictly provides
symptomatic relief. New therapies are needed to provide symptomatic
relief and protection against continued neuronal degeneration to
those who suffer from neurodegenerative diseases such as PD.
[0009] As in HD, there are decreased levels of GM1 in the PD brain
(particularly in the substantia nigra) and administration of bovine
brain-derived GM1 is neuroprotective in animal and cell models of
PD and has both symptomatic effects and slows disease progression
in PD patients. However, due to drawbacks associated with the
administration of animal brain-derived GM1, including the low
ability of systemically administered GM1 to gain access to the
brain, there is a need for new methods of treatment for increasing
GM1 levels in the brain.
SUMMARY OF THE INVENTION
[0010] In accordance with these and other objects, a first
embodiment of an invention disclosed herein is directed to a
composition of matter comprising a viral vector expressing an
exogenous nucleic acid sequence comprising a functional sialidase
Neu3 enzyme and a promoter operably linked to said nucleic acid
sequence. Said composition can suitably be utilized for treatment
of neurodegenerative diseases or disorders to modify the amounts of
GM1 in the brain.
[0011] A method of increasing the amount of GM1 in a brain tissue
comprising administering to a patient an effective amount of an AAV
encoding for sialidase Neu3, to the patient, wherein the sialidase
Neu3 is effective in increasing the amounts of GM1 in the brain
tissue of interest.
[0012] A method of increasing the amount of GM1 in a brain tissue
comprising a concomitant treatment of GM1 and gene therapy
comprising an AAV encoding for sialidase Neu3, wherein the GM1 is
administered to directly increase GM1 levels in the brain and the
AAV is administered to upregulate the production of GM1 through
modification of the sialidase Neu3 so as to modify and stabilize
the levels of GM1 in the brain; and wherein the levels of GM1 in
the brain are thereafter increased as compared to the levels prior
to administration.
[0013] A further embodiment is directed to a method of increasing
the amount of GM1 in a brain tissue comprising a first step of
administering an effective amount of an AAV encoding for sialidase
Neu3 to a patient followed by a concomitant therapy comprising
administration of an AAV to administer a gene encoding for B3Galt4.
Based on experimental data, the gene encoding St3Gal2 (which
converts GM1 to GD1a and GD1b to GT1b) is also downregulated in HD
models and HD fibroblasts. Furthermore, there is some evidence that
St3Gal2 is also downregulated in PD. Therefore, an appropriate AAV
to administer St3Gal2 can also be utilized to increase levels of
GD1a and GT1b which could then be converted into GM1 via AAV
Neu3.
[0014] A further embodiment is directed to a method for treating a
neurodegenerative disease or condition comprising administering an
effective amount of a human compatible engineered adeno-associated
virus (AAV) or lentivirus containing the B3Galt4 sequence under the
control of neuronal specific promoters administered to the patient
with a neurodegenerative disease or condition by intracranial
stereotaxic injunction or by systemic administration.
[0015] A further embodiment is directed to a method to increase
endogenous GM1 ganglioside in the substantia nigra in a PD patient
or in the caudate nucleus, the putamen and/or additional affected
brain regions in an HD patient, by administering an effective
amount of a composition encoding for Neu3, and optional concomitant
administration of shRNA or siRNA directed against St3gal2 to
increase GM1 levels. Through administration of a Neu3-based
therapy, it would be an advantage to try to increase GD1a and GT1b
which are substrates for Neu3 and will help to allow more GM1 to be
made.
[0016] A further embodiment is directed to a method for increasing
GM1 levels in a patient suffering from a neurodegenerative disease
or condition comprising administering to said patient an effective
amount of an shRNA or siRNA for St3gal2, sufficient to increase GM1
levels in said patient with a concomitant therapy comprising
administration of B3Galt4 cDNA to said patient.
[0017] A further embodiment is directed to a method to increase
endogenous GM1 ganglioside in the brain comprising administering
GM1 directly to a patient suffering from a neurodegenerative
disease and administering B3Galt4 cDNA to increase production of
GM1 in the brain; wherein the direct administration provides direct
increase of GM1 in the brain and the B3Galt4 cDNA provides
protective and restorative effects by modulating and increasing the
native production of GM1 in the brain.
[0018] A further embodiment is directed to a method to increase
endogenous GM1 ganglioside in the brain comprising administering
GM1 directly to a patient suffering from a neurodegenerative
disease or condition and administering B3Galt4 cDNA and
concomitantly administering shRNA/siRNA St3gal2 to increase levels
of GM1 in the brain and decrease degradation of GM1; wherein the
direct administration of GM1 increases GM1 directly and the B3Galt4
cDNA and shRNA/siRNA for St3gal2 provide protective and restorative
effects by modulating and increasing the native production of GM1
in the brain.
[0019] A further embodiment is a method of increasing the amount of
GM1 in a brain tissue comprising a concomitant treatment of a
dietary supplement with complex milk lipids (CMLs) having a
concentrated dietary source of GM3, to provide increase levels of
substrate GM3 and GM2 for conversion to GM1 and gene therapy
comprising an AAV or lentivirus encoding for B3Galt4, wherein the
CMLs provide increased substrate to convert GM3 and GM2 to GM1 and
increased expression of B3Galt4 increases conversion of GM2 to GM1
in the brain. The AAV or lentivirus or non-viral vector is
administered to up regulate the production of B3Galt4; and wherein
the levels of GM1 in the brain are thereafter increased as compared
to the levels prior to administration.
[0020] A further embodiment is directed to a composition of matter
suitable for increasing GM1 in the brain comprising a vector
expressing an exogenous nucleic acid sequence comprising a
functional sialidase directed to a Neu3 enzyme, B3Galt4, St3Gal2,
or combinations thereof, and a promoter operably linked to said
nucleic acid sequence. Said composition can suitably be utilized
for treatment of neurodegenerative diseases or disorders to modify
the amounts of GM1 in the brain.
[0021] A further embodiment is directed to a method for treating a
neurodegenerative disease or condition comprising administering an
effective amount of a human compatible engineered non-viral vectors
including liposomes, polymersomes, lipopolyplex, or bolaamphiphile
nanovesicles (that can be targeted to specific neuron subtypes)
containing the Neu3 sequence under the control of neuronal specific
promoters. The same basic strategies for non-viral vectors are also
suitably used for B3Galt4 or St3Gal2 as described herein.
[0022] A further method is directed to treating a neurodegenerative
disease comprising administering to a patient a human compatible
lentivirus encoding for Sialidase Neu3, B3Galt4, or both.
Appropriate strategies may employ use of any one of the
compositions of matter as a medicament for treating a
neurodegenerative disorder or use of said compositions of matter
for increasing GM1 in the brain.
[0023] A method of increasing the amount of GM1 in a brain tissue
comprising a concomitant treatment of a dietary supplement with
complex milk lipids (CMLs) having a concentrated dietary source of
GM3, to provide increase levels of substrate GM3 and GM2 for
conversion to GM1 and gene therapy comprising an AAV encoding for
sialidase Neu3, wherein the CMLs provide increased substrate to
convert GM3 and GM2 to GM1 in the brain and the AAV is administered
to up regulate the production of GM1 through modification of
sialidase Neu3 so as to modify and stabilize the levels of GM1 in
the brain; and wherein the levels of GM1 in the brain are
thereafter increased as compared to the levels prior to
administration.
[0024] A composition of matter comprising an adeno-associated virus
(AAV) or other human compatible virus, encoding the gene for
Sialidase Neu3, B3Galt4, St3Gal2, or combinations thereof, and a
neuron specific promoter, wherein the composition is suitable for
administration to a patient comprising injecting the AAV or other
human compatible virus into the brain by intracranial stereotaxic
injunction; wherein the AAV's encoding for the Sialidase Neu3,
B3Galt4, St3Gal2, or combinations thereof enhance and/or normalize
levels of GM1 in neurons, providing both therapeutic relief and
disease modifying effects in specific areas of the brain relevant
to particular neurodegenerative diseases.
[0025] Therefore, there is a need for new methods of treatment of
neurodegenerative diseases such as PD and HD, including treatments
and mechanisms for regulating or reversing a defect in B3Galt4
expression through administration of B3Galt4 cDNA or for
administering a sialidase Neu3 cDNA, or combinations thereof, to
increase GM1 in the brain.
[0026] Additional features and embodiments will be apparent to one
of ordinary skill in the art upon consideration of the following
detailed description of preferred embodiments and descriptions of
the best mode of carrying out the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1A-B depicts a flowchart showing ganglioside
biosynthetic pathways.
[0028] FIG. 2 depicts an experimental overview of hES cells treated
with MPP.sup.+ and GM1 or sialidase.
[0029] FIGS. 3A and 3B depict the protection/rescue of human
embryonic stem cells from MPP.sup.+ toxicity.
[0030] FIG. 4 depicts striatal uptake of tritiated dopamine
(.sup.3H-DA).
[0031] FIG. 5 depicts immunofluorescent detection of gangliosides
in mouse brain and effects of sialidase on dopamine levels and
number of dopamine neurons.
[0032] FIGS. 6A and 6B depict experimental overviews of certain
mouse studies.
[0033] FIG. 7 depicts the effects of sialidase treatment on
striatal dopamine levels in a PD model.
[0034] FIGS. 8A, 8B, 8C, and 8D depict the effects of sialidase
treatment on substantia nigra neurons in a PD model.
[0035] FIG. 9 depicts AAV-hNeu3 expression in mouse substantia
nigra 2 weeks after administration.
[0036] FIG. 10 depicts neuroprotection in an MPTP mouse PD model
with AAV-hNeu3 administration.
[0037] FIG. 11 depicts symptomatic and disease modifying effects of
GM1 in PD patients.
[0038] FIG. 12 depicts that some gangliosides are reduced in PD
substantia nigra.
[0039] FIG. 13A-B depicts changes in B3Galt4 gene expression SN and
decreased B3Galt4 protein expression in human PD substantia
nigra.
[0040] FIG. 14A-B depicts enhancement of cell death when B3Galt4 is
downregulated.
[0041] FIG. 15A-B depicts downregulation of B3Galt4 in HD models
and HD fibroblasts.
[0042] FIG. 16 depicts neuroprotective effect of AAV-B3Galt4 in the
MPTP mouse PD model.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] The embodiments of the invention and the various features
and advantages thereto are more fully explained with references to
the non-limiting embodiments and examples that are described and
set forth in the following descriptions of those examples.
Descriptions of well-known components and techniques may be omitted
to avoid obscuring the invention. The examples used herein are
intended merely to facilitate an understanding of ways in which the
invention may be practiced and to further enable those skilled in
the art to practice the invention. Accordingly, the examples and
embodiments set forth herein should not be construed as limiting
the scope of the invention, which is defined by the appended
claims.
[0044] As used herein, terms such as "a," "an," and "the" include
singular and plural referents unless the context clearly demands
otherwise.
[0045] The term "increase" is generally used to describe a positive
change in the levels of GM1 as compared to pre-treatment levels in
the brain.
[0046] The term "AAV" means adeno-associated virus.
[0047] The term "B3Galt4" means beta-1,3-galactosyltransferase 4 or
GM1 synthase.
[0048] The term "Sialidase Neu3" means neuraminidase enzyme
Neu3.
[0049] As used herein, the term "about" means plus or minus 5% of
the numerical value of the number with which it is being used.
Therefore, about 50% means in the range of 45%-55%.
[0050] "Administering" when used in conjunction with a therapeutic
means to administer a therapeutic directly to a subject, whereby
the agent positively impacts the target. "Administering" a
composition may be accomplished by, for example, injection, oral
administration, topical administration, or by these methods in
combination with other known techniques. Such combination
techniques include heating, radiation, ultrasound and the use of
delivery agents. When a compound is provided in combination with
one or more other active agents (e.g. other anti-atherosclerotic
agents such as the class of statins), "administration" and its
variants are each understood to include concurrent and sequential
provision of the compound or salt and other agents.
[0051] By "pharmaceutically acceptable" it is meant the carrier,
diluent, adjuvant, or excipient must be compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof.
[0052] "Composition" as used herein is intended to encompass a
product comprising the specified ingredients in the specified
amounts, as well as any product which results, directly or
indirectly, from combination of the specified ingredients in the
specified amounts. Such term in relation to "pharmaceutical
composition" is intended to encompass a product comprising the
active ingredient(s), and the inert ingredient(s) that make up the
carrier, as well as any product which results, directly or
indirectly, from combination, complexation or aggregation of any
two or more of the ingredients, or from dissociation of one or more
of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. Accordingly, the
pharmaceutical compositions of the present invention encompass any
composition made by admixing the viral or non-viral vectors, cDNA
or other delivery vehicles described in the present invention and a
pharmaceutically acceptable carrier.
[0053] As used herein, the term "agent," "active agent,"
"therapeutic agent," or "therapeutic" means a compound,
composition, or viral or non-viral delivery vehicle utilized to
treat, combat, ameliorate, prevent or improve an unwanted condition
or disease of a patient. Furthermore, the term "agent," "active
agent," "therapeutic agent," or "therapeutic" encompasses a
combination of one or more of the compounds of the present
invention.
[0054] A "therapeutically effective amount" or "effective amount"
of a composition is a predetermined amount calculated to achieve
the desired effect, i.e., to inhibit, block, or reverse the
activation, migration, proliferation, alteration of cellular
function, and to preserve the normal function of cells. The
activity contemplated by the methods described herein includes both
medical therapeutic and/or prophylactic treatment, as appropriate,
and the compositions of the invention may be used to provide
improvement in any of the conditions described. It is also
contemplated that the compositions described herein may be
administered to healthy subjects or individuals not exhibiting
symptoms but who may be at risk of developing a particular
disorder. The specific dose of a compound administered according to
this invention to obtain therapeutic and/or prophylactic effects
will, of course, be determined by the particular circumstances
surrounding the case, including, for example, the compound
administered, the route of administration, and the condition being
treated. However, it will be understood that the chosen dosage
ranges are not intended to limit the scope of the invention in any
way. A therapeutically effective amount of composition of this
invention is typically an amount such that when it is administered
in a physiologically tolerable excipient composition, it is
sufficient to achieve an effective systemic concentration or local
concentration in the tissue depending on the form of
administration.
[0055] The terms "treat," "treated," or "treating" as used herein
refer to both therapeutic treatment and prophylactic or
preventative measures, wherein the object is to prevent or slow
down (lessen) an undesired physiological condition, disorder, or
disease, or to obtain beneficial or desired clinical results. For
the purposes of this invention, beneficial or desired results
include, but are not limited to, alleviation of symptoms;
diminishment of the extent of the condition, disorder, or disease;
stabilization (i.e., not worsening) of the state of the condition,
disorder, or disease; delay in onset or slowing of the progression
of the condition, disorder, or disease; amelioration of the
condition, disorder, or disease state; and remission (whether
partial or total), whether detectable or undetectable, or
enhancement or improvement of the condition, disorder, or disease.
Treatment includes prolonging survival as compared to expected
survival if not receiving treatment.
[0056] In various animal models of PD, systemic administration of
the a-series ganglioside, GM1, protects dopaminergic (DAergic)
neurons from degeneration and restores function to damaged neurons.
In a recent clinical trial, administration of GM1 to PD patients
resulted in symptomatic improvement and slowed the progression of
symptoms. Ganglioside deficiencies and imbalances have been
observed in aging and have been suggested to occur in
neurodegenerative diseases and these changes may exist in the PD
brain. There may be a specific GM1 deficiency and possible decrease
in other gangliosides in PD that contributes to neurodegeneration
of the nigrostriatal pathway. To explore this possibility, we
examined the expression of GM1 (as well as other a- (ex., GD1a) and
b- (ex., GD1b and GT1b) series gangliosides) as well as mRNA
expression for some of enzymes responsible for ganglioside
biosynthesis in the PD brain. We have also examined expression of
ganglioside-related genes in dopamine neurons isolated from post
mortem PD brain and normal control brain.
[0057] The more highly expressed gangliosides in the brain are GM1,
GD1a, GD1b, GT1b, and GD3. GD3 is a potential mediator of apoptosis
whereas GM1 has been shown to be broadly neuroprotective in a
variety of systems and models. Preclinical studies showed that GM1
treatment in various PD models resulted in significant behavioral
and biochemical recovery including protection/rescue of damaged
dopamine (DA) neurons and increased DA levels in the striatum. A
recent randomized, controlled, delayed start clinical study showed
that in PD patients, GM1 use for the first 24 weeks of the study
was superior to placebo for improving motor symptoms and that
extended GM1 use (up to 120 weeks) resulted in a sustained clinical
benefit and lower than expected symptom progression. These data
suggest that enhancing GM1 levels in PD may be disease modifying.
Although GM1 administration has promise as a treatment for PD, it
is difficult to source, is currently available only as a natural
product extracted and purified from animal brains, and has low
ability to cross the blood brain barrier.
[0058] The reasons why GM1 treatment is so effective clinically in
PD are not completely clear but it is possible that PD is
characterized in part by a disorder of glycolipid
biosynthesis/metabolism, in particular, a GM1 deficiency disorder,
and that GM1 administration represents GM1 replacement therapy. The
pathophysiology of the disease may involve alterations in the
pathways involved with ganglioside biosynthesis and/or metabolism
in the PD brain.
[0059] An alternative therapeutic approach to administering GM1 is
to increase endogenous GM1 levels in the brain. One method for
enhancing GM1 levels in the brain involves the manipulation of
ganglioside degradation by sialidases. Sialidases hydrolyze sialic
acid linkages on gangliosides and can degrade complex gangliosides
(and GD3) while increasing GM1 (where the sialic acid linkage is
not susceptible to the sialidase). Specifically, sialidases
catalyze the removal of .alpha.-linked sialic acid residues from
carbohydrate groups of gangliosides. Thus, gangliosides GD1b, GT1b,
and GD1a can be converted to GM1 and the potentially apoptogenic
GD3 can be degraded.
[0060] The human sialidase Neu3 has unique substrate specificity
(i.e., degrades complex gangliosides GD1a, GT1b, and GD1b into GM1,
does not cleave internal sialic acids on GM1 and GM2, and
hydrolyzes GD3) and associates with plasma membranes. Neu3 is
active toward plasma membrane gangliosides where it can modify the
ganglioside content, specifically increasing GM1 levels and
potentially modifying important functional membrane events such as
cell signaling. Neu3 also possesses anti-apoptotic properties.
Transfection of the Neu3 gene into cancer cells up-regulated Neu3
and inhibited apoptosis, increased Bcl-2 protein, and decreased
caspase-3 expression. In contrast, silencing Neu3 expression
induced apoptosis without specific stimuli and was accompanied by
decreased Bcl-xL (pro-survival protein) expression. Our studies
have shown that this process can be stimulated in vivo and it
confers neuroprotection in PD models similar to that achieved with
administration of GM1.
[0061] Indeed, the most highly expressed gangliosides in brain are
GM1, GD1a, GD1b, GT1b, and GD3. While GM1 is broadly
neuroprotective in a variety of systems and models, GD3 is a
potential mediator of apoptosis. The therapeutic method described
in this invention will simultaneously decrease GD3 levels (a
potential mediator of toxicity) and/or increase GM1 levels
(potentially neuroprotective), providing a superior neuroprotective
effect. However, any increase in GM1 levels, even without
concomitant depreciations in GD3 levels, provides a neuroprotective
effect.
[0062] In HD models, it has been shown that intracranial
administration of bovine brain-derived GM1 resulted in normalized
motor function, a concomitant normalization of striatal dopamine
levels (critical to normal striatal function), and phosphorylation
of mHtt at serine residues 13 and 16 that attenuated mHtt toxicity.
These data suggest that enhancing GM1 levels in HD may also be
disease modifying.
[0063] Therefore, because each of HD and PD exhibit decreases in
GM1 ganglioside levels, mechanisms to support GM1 levels or
increase GM1 levels will provide protective and stabilizing effects
in the brain.
[0064] Unfortunately, no drug has yet been developed that has an
unequivocal positive effect on disease progression. What is needed
is a new treatment for PD that slows the degeneration of substantia
nigra dopamine neurons and thus slows, arrests, or reverses the
progression of the disease.
[0065] GM1 levels can be increased in the brain in several
different ways. A first mechanism is to simply administer GM1 to
the patient to increase GM1 levels in the affected tissues. This
mechanism requires systemic administration of large amounts of GM1
due to the poor ability of GM1 to transport into the brain, has
short term benefits of immediate increase of the levels, but
suffers from some long term issues, including practical and safety
concerns regarding sourcing GM1 from animal brains, the low
bioavailability/penetrance of systemically administered GM1 across
the blood brain barrier, and no oral bioavailability requiring
administration by injection potentially multiple times per day or
chronic intracerebral infusion. Therefore, chronic administration
of GM1 alone may be difficult and not ideal, based on currently
available methods.
[0066] Therefore, a preferred embodiment relates to administering a
gene therapy designed to increase expression of the human sialidase
(neuraminidase) enzyme Neu3, which removes sialic acid residues
from complex polysialogangliosides converting them into GM1.
Preliminary studies have shown that administration of sialidase
enzyme directly to the brain in an animal model of PD has a
neuroprotective effect similar to administration of GM1. This
mechanism will increase GM1 levels in the targeted tissue
(substantia nigra and other brain tissues) and provide a
neuroprotective effect similar to that obtained in clinical trials
performed in which GM1 was administered to PD patients.
[0067] Sialidases, in particular, sialidase Neu3, hydrolyze sialic
acid linkages on gangliosides and can degrade complex gangliosides
(and GD3) converting them into GM1. Thus, as described above, GD1b,
GT1b, and GD1a, are partially converted to GM1 and the potentially
apoptogenic GD3 is partially degraded. Thus, application of Neu3
gene therapy will reshape the glycolipid composition of the cell,
resulting in a beneficial variation in plasma membrane gangliosides
and together with anti-apoptotic effects, will be neuroprotective.
Accordingly, the gene therapy encoding for Neu3, provides for a
mechanism to partially convert complex polysialogangliosides in the
brain into GM1.
[0068] The sialidase can be administered via a gene therapy
designed to increase expression of GM1 wherein human compatible
engineered AAV containing the Sialidase Neu3 sequence under an
appropriate promoter is administered. Administration of the AAV is
preferentially administered via intracranial stereotaxic injection.
In other suitable embodiments, administration of the AAV and the
genes encoding for the sialidase Neu3 gene may be administered via
other mechanisms known to one of ordinary skill in the art.
[0069] Actions of Sialidase
[0070] There are a number of different sialidases with somewhat
different actions. For example, sialidase C purified from
Clostridium perfringens (CPS) preferentially cleaves a2-3 linkages,
has comparatively little activity at a2-8 linkages and should
result primarily in a conversion of GD1a to GM1 (see below).
Sialidase V purified from Vibrio cholerae (VCS) primarily cleaves
a2-8 linkages and terminal a2-3 linkages and should result in a net
decrease in GD1a and b-series gangliosides GD1b and GT1b and
increased GM1. VCS should also degrade GD3. Therefore, these
increases in endogenous GM1 levels (and decreases in potentially
damaging GD3 levels) will be neuroprotective against dopamine
neuron damage and death from a variety of causes including
MPTP/MPP.sup.+-mediated DA neuron toxicity as well as DA neuron
toxicity mediated by a wide variety of other trigger mechanisms.
Indeed, gangliosides GD3, GM3, GD1a and GD1b, but not GM1 or GM2,
are good substrates for sialidase Neu3 for increasing endogenous
GM1 levels.
[0071] Indeed, FIGS. 1A and 1B depict diagrams of the ganglioside
biosynthetic pathways, and depicts several mechanistic ways in
which GM1 can be increased through modification of the GM1
biosynthetic pathways. In FIG. 1B, the underlined sialic acids are
removed by the action of the sialidase, thus converting those
gangliosides into GM1.
EXAMPLES
[0072] Human Embyonic stem cell (hES) culture: Human stem cells
were differentiated into a dopaminergic (DAergic) phenotype using a
well characterized method (Iacovitti et al. 2007 as modified in Cai
et al., 2013).
[0073] FIG. 2 depicts a brief diagram of the experimental overview.
Depicted on the top is the material administered and on the bottom
is the number of hours.
[0074] Rescue of DAergic hES cells: hES cells were plated onto 24
well plates. Cells were pretreated with sialidase, GM1 or standard
media for 2 hrs and were then exposed to the mitochondrial toxin
MPP.sup.+. Twenty-four hours later, media containing toxin was
removed and fresh media containing the appropriate concentration of
rescue agent was added. Two days later, cells were fixed, processed
for tyrosine hydroxylase (TH) immunocytochemistry and the number of
TH immunopositive cells were counted.
[0075] For in vivo studies, sialidases were prepared in artificial
cerebral spinal fluid (aCSF) and loaded into Alzet osmotic pumps
(model 2004). Brain infusion cannulae were attached to the pumps to
allow for intracerebroventricular (ICV) administration of
sialidases or vehicle. Brain infusion cannulae were placed in the
third ventricle and osmotic pumps were implanted into C57B16 male
mice (age 7-10 weeks). One week after surgery, sub-acute MPTP
administration (2 injections 20 mg/kg s.c. for 5 days) was
initiated. Mice were euthanized 2 weeks after the final MPTP
injection. In a separate group of animals, GM1 was administered
daily (30 mg/kg i.p.) beginning 24 hrs after the final MPTP
injection and continuing for two weeks.
[0076] Some animals receiving sialidase or aCSF alone were
euthanized 12 days post-surgery to examine the effects of sialidase
treatment on DA uptake, to examine if sialidase would interfere
with DA uptake and inhibit the access of MPTP into DA neurons.
Whole striatal homogenates were exposed to [.sup.3H] DA for 3
minutes in the presence or absence of mazindol to examine the
influence of sialidase treatment on DA uptake.
[0077] A the conclusion of the study, the striatum contralateral to
the implant was collected to analyze striatal catecholamine and
metabolite levels by high performance liquid chromatography (HPLC)
according to previously published methods (Kidd and Schneider
2011). Briefly, samples were sonicated in perchloric acid and the
soluble fraction was separated using MDTM mobile phase (11%
acetonitrile, pH 3.0) on an ESA Coulochem III HPLC system with
electrochemical detection.
[0078] Sections were cut frozen at 30 .mu.m on a sliding microtome.
Every third section through the rostro-caudal extent of the
substantia nigra (SN) was processed for TH, the adjacent section
was stained with cresyl violet and remaining sections were used for
detection of gangliosides. All sections were washed in PBS,
(endogenous peroxidase activity was blocked using peroxide for
IHC), blocked, and exposed to primary antibody overnight. Sections
were then exposed to biotinylated secondary, avidin biotin complex
and developed for fluorescence microscopy.
[0079] Microbrightfield StereoInvestigator was used to estimate
numbers of TH immunopositive and cresyl violet stained cells in the
SN by unbiased stereology, as described by us previously (Kidd and
Schneider 2011).
[0080] FIGS. 3A and 3B depict the protection/rescue of human
embryonic stem cells from MPP.sup.+ toxicity. A) The number of
tyrosine hydroxylase immunopositive (TH.sup.+) cells was reduced as
a result of MPP.sup.+ treatment. GM1 treatment prevented cell loss
at the 5 and 10 .mu.M doses of MPP.sup.+. *p<0.01 vs control,
{circumflex over ( )}p<0.05 vs respective MPP.sup.+ only,
**p<0.01 vs respective MPP.sup.+ only. B) Sialidase from Vibrio
cholerae (VCS) was also able to protect/rescue cells from MPP.sup.+
toxicity. {circumflex over ( )}p<0.01 vs control; **p<0.01 vs
respective MPP.sup.+ only.
[0081] In mouse studies as depicted in FIG. 4, striatal uptake of
tritiated dopamine (.sup.3H-DA) was examined. FIG. 6A depicts an
experimental overview of a dopamine uptake study. Striatal
homogenates were incubated with .sup.3H-DA in the presence
(non-specific) or absence (total) of the DA uptake inhibitor
mazindol (Maz). Mazindol effectively blocked total DA uptake;
sialidase treatment did not, indicating that the sialidase does not
interfere with the mechanism of uptake of MPP.sup.+. FIG. 4 further
shows that specific uptake of DA in the striatum was not inhibited
by intracerebral administration of sialidase to mice for 12
days.
[0082] FIG. 5 depicts immunofluorescent detection of gangliosides
in mouse striatum. FIG. 5 depicts striatal sections showing an
increase in GM1 and a decrease in GT1b, GD1a, as a result of
sialidase treatment.
[0083] FIG. 6B depicts a mouse study from days 0-26 wherein days
7-12 include the animals being subjected to MPTP, the results of
which are depicted in FIG. 7.
[0084] FIG. 7--Effects of sialidase on striatal dopamine levels by
HPLC. MPTP treatment resulted in significant decreases in striatal
dopamine (DA). In mice that received sialidase infusion for 7 days
prior to MPTP and then for an additional 21 days, there was a
significant sparing of striatal DA levels compared to animals that
received control (aCSF) infusions. **p<0.01 vs aCSF-MPTP
[0085] FIG. 8--Effects of sialidase treatment on substantia nigra
(SN) cells. Stereology was used to estimate the number of
dopaminergic cells in the substantial nigra. Dopaminergic cells
(TH.sup.+) with an identifiable nucleus were counted at 100.times.
magnification. Nissl cell counts (number of cresyl violet stained
cells) were obtained using the same counting regions of the SN from
an individual animal as used in adjacent TH.sup.+ sections (FIGS.
8A, 8B: ***p<0.01 vs aCSF-MPTP; {circumflex over ( )}p<0.001
vs aCSF-MPTP). Sub-acute MPTP treatment resulted in significant
decreases in TH.sup.+ and Nissl-stained cell numbers. GM1 and
sialidase treatment had similar protective effects on SN dopamine
neurons (FIGS. 8C (TH.sup.+ cells), 8D (cresyl violet stained
cells): **p<0.001 vs GM1; {circumflex over ( )}p<0.0001 vs
GM1; .about.p<0.001 vs GM1).
[0086] FIG. 9--AAV-hNeu3 expression in mouse substantia nigra (SN)
2 weeks after administration. An injection of AAV-hNeu3
(1.7.times.10.sup.10 GC in 1 .mu.l) was placed just dorsal to the
substantia nigra (SN) on one side of the brain. Two weeks later,
the animals were euthanized and the SN on each side was dissected
and frozen. RNA was extracted from the tissue samples and the
samples were run on RT-PCR to assess expression of the hNeu3
transgene.
[0087] FIG. 10--Neuroprotection in an MPTP mouse PD model with
AAV-hNeu3 administration. AAV-hNeu3 was administered by injection
(1.7.times.10.sup.10 GC in 1 ul) just dorsal to the substantia
nigra (SN) on one side of the brain. Two weeks later, MPTP was
administered twice daily for 5 consecutive days. Two weeks after
the last MPTP injection, the animals were euthanized by
transcardial perfusion and the brains sectioned and labeled for
visualization of green fluorescent protein (used as a tag on the
AAV to enable localization of the virus in tissue) and tyrosine
hydroxylase (to label dopamine neurons). On the side of the brain
where AAV-hNeu3 was administered (ipsilateral), there were more
dopamine neurons in the SN than compared to the opposite side of
the brain that did not receive AAV-hNeu3 (contralateral).
[0088] Accordingly, the examples and studies identify that human
embryonic stem (hES) cells, when differentiated into dopaminergic
neurons, are susceptible to MPP.sup.+ toxicity. However, there are
mechanisms that exist that can mitigate the effects of MPP.sup.+
toxicity. Indeed, MPP.sup.+ toxicity in hES cells is mitigated by
both GM1 and 0.005 U/mL VCS. Therefore, treatment with a sialidase,
such as sialidase Neu3 that increases the amounts of GM1 expression
in the striatum and the substantia nigra will provide
neuroprotective effects.
[0089] Furthermore, the experiments identify that sialidase
treatment does not alter MPP.sup.+ uptake in vivo and that lack of
MPP.sup.+ uptake is not the mechanism for neuroprotection from
sialidase administration. Conversely, sialidase treatment results
in changes in ganglioside expression, resulting in an increase in
GM1 expression, in both the striatum and substantia nigra. At least
one concentration of sialidase enzyme had a sparing effect on SN
dopaminergic neurons. However, an effective amount of sialidase,
administered to an animal, provided protective effects in most
circumstances. Indeed, some sialidase treatments were as effective
as GM1 administration on sparing striatal dopamine levels and SN
dopamine neurons.
[0090] These data show that increasing endogenous GM1 levels
through the use of sialidase enzymes can have significant
protective/rescue effects on dopamine neurons, and at least in some
instances, to the same extent as administration of GM1 ganglioside.
Since GM1 administration is limited based on the difficulty in
sourcing the material and on low bioavailability, other suitable
materials are important for continuing to learn about and develop
mechanisms to otherwise increase GM1 levels in the brain.
Therefore, sialidase treatment may be as effective as systemic GM1
administration in sparing striatal DA levels and SN neurons and may
be an alternative to systemic GM1 administration as a potential PD
disease modifying strategy.
[0091] An embodiment comprises a therapeutic treatment comprising a
composition of matter such as a pharmaceutical composition
comprising a gene therapy vector expressing an exogeneous nucleic
acid sequence comprising: (1) a nucleic acid sequence encoding a
functional Sialidase Neu3 enzyme; (2) a promoter operably linked to
said nucleic acid sequence; and (3) wherein the composition of
matter is effective at increasing GM1 in the brain.
[0092] Indeed, appropriate methods of therapeutic treatment
comprising administering the sialidase enzyme to a patient having a
neurodegenerative disorder is suitable to prove protective effects
through increasing the levels of GM1 in the brain thereby providing
protective elements of GM1 and reducing or reversing the course of
neurodegenerative disease, such as PD, HD or other known
neurodegenerative diseases and conditions that are or may be
affected by GM1. In certain embodiments, the sialidase enzyme is
appropriately delivered through a composition comprising an AAV
encoding for sialidase Neu3. However, other suitable vectors are
provided and described herein.
[0093] Indeed, it may be further advantageous to provide a
therapeutic treatment comprising concomitant administration of GM1
and a gene therapy comprising an AAV encoding for a sialidase
enzyme, Neu3, wherein the direct administration of GM1 provides
protective effects by increasing GM1 directly and the gene therapy,
through increase of sialidase enzyme Neu3 administration can
provide mechanistic changes in the brain cells to protective
effects through increasing the levels of GM1 in the brain thereby
providing protective elements of GM1 and reducing or reversing the
course of neurodegenerative disease, such as PD, HD or other known
neurodegenerative diseases and conditions that are or may be
affected by GM1.
[0094] In appropriate settings, the compositions described herein
and throughout the embodiments may be advantageously used to treat
neurodegenerative diseases or disorders, wherein use of the
composition generates an increase in GM1 in the brain. In certain
embodiments, use of a composition comprising a viral vector for
Sialidase Neu3 as a medicament for treatment of a neurodegenerative
disease or disorder is appropriate.
[0095] A further therapeutic strategy may utilize the same pathway
identified in FIG. 1 but approach the modification or increase of
GM1 from a different aspect. In essence, the pathway for GM1 via
the arrows depicts a natural flow, wherein GM2 is converted by
B3Galt4 into GM1, and GM1 then converted by St3Gal2 into GD1a, or
GT1a. The sialiadase Neu3 strategy increases GM1 levels by
converting the downstream a-series polysialoganglioside GD1a and
the b-series polysialogangliosides GD1b and GT1b into GM1. However,
a further strategy for increasing GM1 levels would be to increase
GM1 directly by correcting a possible defect or error in the
production of GM1 from GM2 due to decreased expression of the GM1
synthase enzyme, B3Galt4. This could be achieved through B3Galt4
gene replacement.
[0096] Research suggests that in the substantia nigra in the PD
brain, and particularly in isolated dopamine neurons, there is a
decreased expression of the mRNA for the enzyme B3Galt4
(beta-1,3-galactosyltransferase 4)(AKA GM1 synthase), which
contributes to decreased GM1 ganglioside levels in PD substantia
nigra, which in turn, supports the degeneration of dopamine
neurons. Unfortunately, no drug has yet been developed that
unequivocally has a positive effect on disease progression. What is
needed is a new treatment for PD that slows the degeneration of
substantia nigra dopamine neurons and thus slows, arrests, or
reverses the progression of the disease.
[0097] In addition to PD and HD, other neurodegenerative diseases
such as Alzheimer's disease and ALS also suffer from similar GM1
reduction resulting in damage. Furthermore, several peripheral
neuropathies including, but not limited to: diabetic neuropathy,
chemotherapy-induced neuropathy such as "chemo-brain," traumatic
brain injury, acute spinal cord injury, stroke and retinopathies
also may have similar patterns of damage resulting from low GM1 and
would benefit from the treatment suggested herein. Alteration in
GM1 biosynthesis may be a mechanism common to a wide variety of
neurodegenerative disorders that may increase the vulnerability of
neurons to degenerating in response to a variety of stressors.
[0098] In HD models and in HD fibroblasts, it has been shown that
B3Galt4 mRNA is downregulated, resulting in decreased levels of
GM1. It has also been shown in HD models that intracranial
administration of GM1 resulted in normalized motor function, a
concomitant normalization of striatal dopamine levels (critical to
normal striatal function), and phosphorylation of mHtt at serine
residues 13 and 16 that attenuated mHtt toxicity. These data
suggest that enhancing GM1 levels in HD may be disease
modifying.
[0099] Therefore, because each of HD and PD exhibit decreased GM1
ganglioside levels and defects in B3Galt4 expression, mechanisms to
support GM1 levels or increase GM1 levels or correct the B3Galt4
defect will provide protective effects in the brain.
[0100] Furthermore, because of the significant impediments to the
therapeutic delivery of GM1 in neurodegenerative disease, which
include the possible need for chronic intra-cranial infusion or
chronic systemic injection, practical and safety concerns regarding
sourcing GM1 from animal brains, low bioavailability/penetrance of
systemically administered GM1 across the blood brain barrier, and
no oral bioavailability requiring administration by injection
potentially multiple times per day or by continuous intracranial
infusion, modification or increase of GM1 must be mediated through
alternative means.
[0101] Accordingly, a more efficient way to increase GM1 levels
will be by modifying the cell's own ganglioside biosynthetic
capacity utilizing viral or non-viral mediated gene therapy to
increase expression of B3Galt4, the enzyme responsible for GM1
synthesis and deficient in PD and HD. Therefore, a preferred
embodiment of the invention is particularly related to a method for
treating neurodegenerative disease such as PD or HD that is
expected to have disease-modifying effects of slowing the
progression of the disease comprising increasing the expression of
B3Galt4 in the patient.
[0102] A preferred mechanism uses administration of a composition
comprising GM1 synthase cDNA in an expression vector to increase
expression of the enzyme responsible for the direct biosynthesis of
GM1. This will increase endogenous levels of GM1 in substantia
nigra neurons (in PD) or striatal or cortical neurons (in HD) and
leads to the same disease modifying effect as is seen with
administration of GM1 ganglioside. This may be achieved alone or in
combination with administration of substances intended to increase
levels of the precursor(s) for GM1.
[0103] Using adeno-associated virus (AAV) encoding these genes
under a neuron specific promoter, we will enhance endogenous GM1
expression following a single intracranial administration. A
preferred embodiment comprises a composition of matter such as a
pharmaceutical composition comprising a gene therapy vector
expressing an exogenous nucleic acid sequence comprising: (1) a
nucleic acid sequence encoding a functional B3Galt4 enzyme; (2) a
promoter operably linked to said nucleic acid sequence; and (3)
wherein the composition of matter is effective at increasing GM1 in
the brain.
[0104] Accordingly, a proposed method comprises modifying the
cell's own ganglioside biosynthetic capacity utilizing viral
mediated gene therapy to increase expression of B3Galt4. Indeed, a
human compatible engineered adeno-associated virus (AAV) containing
the B3Galt4 sequence under the control of neuronal specific
promoters can be administered to the patient with neurodegenerative
disease by intracranial stereotaxic injunction. The AAV's
containing the B3Galt4 sequence would then enhance and/or normalize
levels of GM1 in neurons, providing both therapeutic relief and
disease modifying effects. Furthermore, advances in AAV
technologies now provide the potential for systemic administration
of AAV's with neuronal targeting in juveniles or adults, allowing
for non-surgical treatment of HD in newborns or in at-risk
individuals which may further delay/inhibit the occurrence of the
disease.
[0105] In further embodiments, other forms of gene therapy or other
mechanisms of administration are suitable. For example, one of
several human compatible viral vectors are suitable for
administering the genes of interest. This includes, but is not
limited to such viral vectors as lentiviruses. Furthermore, it may
be beneficial in certain embodiments to use a lentiviral vector to
transmit a B3Galt4 sequence, a sialidase Neu3 sequence, or both to
a patient.
[0106] In further embodiments non-viral vectors may be used for
administering the genes of interest. These include use of
liposomes, polymersomes, lipopolyplex, or bolaamphiphile
nanovesicles (that can be targeted to specific neuron subtypes).
Therefore, certain targeted approaches can be utilized and
formulated for specific intra cranial injection or through IV.
[0107] In a further embodiment, in conjunction with B3Galt4 gene
therapy, the levels of GM1 can be modified through a further
pathway wherein the GM1 levels are not upregulated, but the
breakdown of GM1 into other gangliosides can be reduced, therefore
increasing the levels of GM1. In one pathway, shRNA/siRNA is used
to decrease expression of St3gal2 (ST3 beta-galactoside
alpha-2,3-sialyltransferase 2 "SIAT4B"). St3gal2 adds sialic acid
to the terminal galactose of Gal.beta.1-3GalNAc-terminated
glycolipids, such as GM1 and GD1b, to synthesize GD1a and GT1b.
Down-regulation of the gene for St3gal2 and inhibition of
production of this enzyme results in decreased levels of GD1a and
GT1b with a concomitant increase in GM1. This has been shown to
occur in St3gal2-null mouse brain (Sturgill et al., 2012).
[0108] Therefore, a further proposed embodiment is directed to a
composition to increase endogenous GM1 ganglioside levels in the
substantia nigra by reversing a defect in B3Galt4 through
administration of B3Galt4 cDNA, alone, or in combination with
shRNA/siRNA St3gal2, which will further increase GM1 levels,
wherein said composition of matter comprises a gene therapy vector
expressing an exogeneous nucleic acid sequence comprising: (1) a
nucleic acid sequence encoding a functional B3Galt4 enzyme; (2) a
promoter operably linked to said nucleic acid sequence; (3)
shRNA/siRNA St3gal2; and (4) wherein the composition of matter is
effective at increasing GM1 in the brain. Wherein the shRNA/siRNA
St3gal2 may be operably linked to the therapeutic vector or
provided as a separate medicament and administered concurrently
with the therapeutic vector.
[0109] Therefore, a further proposed embodiment is directed to a
method to increase endogenous GM1 ganglioside levels in the
substantia nigra by reversing a defect in B3Galt4 through
administration of B3Galt4 cDNA, alone, or in combination with
shRNA/siRNA St3gal2, which will further increase GM1 levels.
[0110] In treatment of PD, principal administration is direct
application of the gene therapy to the substantia nigra. While the
treatment is somewhat invasive, PD patients currently receive brain
surgery from implantation of deep brain stimulation electrodes.
Furthermore, there have been several gene therapy trials in PD
including AAV2-GDNF and ProSavin that required surgery. Other
suitable routes of administration are discussed throughout this
application, wherein the appropriate dose and formulation can be
modified by one of ordinary skill in the art.
[0111] In certain embodiments, use of a composition comprising a
viral vector containing cDNA for B3Galt4 as a medicament for
treatment of a neurodegenerative disease or disorder is
appropriate. Alternatively, use of a medicament comprising shRNA or
siRNA st3gal2 is appropriate for treatment of a neurodegenerative
disease or disorder, or use of a combination of medicaments or
therapeutics as described herein.
EXAMPLES
[0112] Tissue: Post-mortem tissue from PD patients and age matched
controls were obtained from the Human Brain and Spinal Fluid
Research Center (Los Angeles, Calif.) or the NICHD Brain and Tissue
Bank at the University of Maryland.
[0113] Fresh frozen tissue was paraffin embedded and sectioned at
10 .mu.m. Sections were briefly fixed in cold 80% acetone prior to
neutral red staining prior to laser capture microdissection (LCM).
For LCM we used the PixCell II Laser Capture Microdissection
instrument (Arcturus Engineering, Mountain View, Calif.). Only DA
neurons in the SN with a defined nucleus and containing
neuromelanin were sampled. RNA was extracted and RNA amplicon
libraries were generated for use with a custom AmpliSeq panel
(focused on glycomics and trophic factors) and Next-Gen sequencing
using the Ion Personal Genome Machine (PGM) System
[0114] Quantitative PCR Analysis:
[0115] RNA was extracted from fresh frozen samples using the Qiagen
miRNeasy kits according to manufacturer's instructions for use on
the QIAcube system. Samples were converted to cDNA using the Qiagen
Omniscript RT kit and oligo dT primer. Real-Time PCR was performed
on cDNA using the Roche LightCycler 480 with Roche LightCycler 480
SYBR Green I Master. 25 ng of cDNA was used per PCR reaction and
all samples were analyzed in triplicate. The reaction conditions
were as follows: B3galt4--95.degree. C. for 10 minutes and 45
cycles of 95.degree. C. for 15 s and 60.degree. C. for 1 minute. To
confirm specificity of amplification the products were subjected to
a melting curve analysis at the end of the final annealing period.
To analyze the PCR data the .DELTA..DELTA.C.sub.t method was used
to calculate fold-regulation of glycosyltransferase mRNA expression
relative to B2M (housekeeping gene) expression.
[0116] FIG. 11--GM1 treatment had an early symptomatic effect and a
later disease modifying effect on PD. The mean (.+-.SE) change from
baseline in Early-start (randomized to receive GM1 at start of
study) and Delayed-start (randomized to receive placebo at start of
study) study subjects and in a standard-of-care comparison group
(no interventions). The dashed vertical line at week 24 indicates
the end of study Phase I. After that, all subjects received GM1.
The dashed vertical line at week 120 indicates the end of study
Phase II. The horizontal dashed line indicates baseline level. An
increase of score indicates symptom worsening; a decrease in score
indicates symptom improvement. *=p<0.0001 Early-start vs.
Delayed-start; {circumflex over ( )}=p<0.05 Early-start vs.
Delayed-start
[0117] FIG. 12 depicts that Gangliosides are reduced in Parkinson's
disease substantia nigra. Thin layer chromatography data from the
SN of 3 PD cases and 4 age-matched controls showing decreased
expression of major brain gangliosides GM1, GD1a, GD1b, and GT1b.
Therefore, modification of these major brain gangliosides via the
mechanisms described herein, leads to neuroprotective effects for
patients suffering from theses neurodegenerative diseases.
[0118] FIGS. 13A and B depict that B3Galt4 gene and protein
expression is significantly altered in PD diseased brains. FIG. 13A
depicts about a two fold reduction of gene expression in PD brain
SN versus age matched control. FIG. 13B further depicts the B3Galt4
Protein Expression in the substantia nigra in PD brain.
[0119] FIGS. 14A and B depict that B3Galt4 siRNA decreases GM1
expression in SK-N-SH Cells. Therefore, and as depicted in FIG.
14B, the downregulation of B3Galt4 results in Enhancement of cell
death when B3Galt4 is downregulated. This again leads to the
conclusion that appropriate modulation of B3Galt4 can reduce the
occurrence of cell death or otherwise stabilize cell death to
prevent or slow the progression of disease.
[0120] FIGS. 15A and B depict the Downregulation of B3Galt4 in HD
models and HD fibroblasts. Therefore, appropriate regulation of
B3Galt4, as mediated by the compositions and therapeutic methods or
uses of the compositions described herein, can mediate the
downregulation of B3Galt4 to reduce the neurodegenerative effects
seen in the neurodegenerative diseases.
[0121] FIG. 16.--depicts side by side images of the contralateral
and ipsilateral brain images that identify the significant
Neuroprotective effect of AAV-B3 Galt4 in the MPTP mouse PD model.
The B3Galt4 was administered on just one side of the brain, and
wherein the side of the brain where the B3Galt4 was administered,
there were more neurons compared to the opposite side of the brain
that did not receive the B3Galt4.
[0122] GM1 levels appear to be reduced in the substantia nigra of
Parkinson's patients even in cells still expressing TH.
Glycosyltransferase gene expression is altered in the substantia
nigra in Parkinson's patients relative to aged matched controls.
Similar gene expression changes also occur in HD brain and in the
brain in other neurodegenerative diseases.
[0123] Therefore, the present results suggest that there may be a
dysregulation of ganglioside biosynthesis in PD (and HD) and that
administration of GM1 to PD patients (and in animal and cell models
of HD) may provide a clinically important increase in GM1 levels in
brain sufficient to overcome a PD (HD)-related GM1 deficit
resulting in a neuroprotective/neurorestorative effect.
[0124] Because GM1 has proven to have neuroprotective effects in
certain neurodegenerative diseases, increase of GM1 proves critical
for protecting and improving the life of those who suffer from such
diseases. Described herein are several approaches to increasing GM1
in the brain. Where all the strategies may work individually, so to
may a combination therapy be relevant for appropriate treatment,
and therapies for patients who suffer from these neurodegenerative
disorders. Therefore, it is appropriate to generate a combination
therapeutic composition that comprises both treatments to correct
the B3Galt4 gene and also for increasing the Sialiadase Neu3. Such
composition may comprise a single viral or non-viral strategy that
includes vectors for each of the strategies, or, a composition may
comprise separate vehicles for administering the two different
strategies. Furthermore, combined composition may further be
administered with additional strategies as described throughout
this document.
[0125] Further methods may employ two separate compositions
administered concurrently in a single medicament, or concurrently
in time, with two or more separate compositions or therapeutics
administered separately, so that the effects are concurrently seen
in the patient.
[0126] For example, an embodiment provides for gene therapy,
including use of viral vectors containing the sialidase Neu3
sequence of interest. For example, a particular viral vector
includes the use of lentiviruses containing the sialidase Neu3
sequence. In certain embodiments, the gene therapy can also include
the B3Galt4 sequence alone, or together with sialidase Neu3. In
certain embodiments, the gene therapy can also include the St3Gal2
sequence alone, or together with sialidase Neu3. Appropriate viral
vectors include lentiviral vectors, retroviruses, adenoviruses,
adeno-associated viruses; further more suitable non-viral vectors
may also be implemented in certain embodiments.
[0127] Therefore, certain embodiments are directed to a concomitant
therapy of administering a gene therapy encoding for the human
sialidase enzyme Neu3 to increase GM1 levels and decrease GD3
levels in the brain, combined with a gene therapy designed to
increase expression of GM1 wherein human compatible engineered AAV
containing B3Galt4 sequence under an appropriate promoter is
administered via intracranial stereotaxic injection. The
combination seeks to increase GM1 production through effects on
different GM1 biosynthetic pathways. The B3Galt4 increases low
levels of B3Galt4 in PD brain to enhance conversion of GM2 into GM1
(and GD1b from GD2), whereas Neu3, when upregulated through gene
therapy, hydrolyzes sialic acid linkages on downstream
polysialogangliosides (including GD1b) to partially convert them to
GM1 to increase GM1 levels in the brain. The St3Gal2 increases low
levels of St3Gal2 in PD brain to enhance conversion of GM1 into
GD1a and GD1b into GT1b, whereas Neu3, when upregulated through
gene therapy, hydrolyzes sialic acid linkages on GD1a and GT1b to
partially convert them to GM1 to increase GM1 levels in the
brain.
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