U.S. patent application number 17/178972 was filed with the patent office on 2021-08-26 for generating gabaergic neurons in brains.
The applicant listed for this patent is The Penn State Research Foundation. Invention is credited to Gong Chen, Yuchen Chen, Ziyuan Guo, Zifei Pei, Zheng Wu.
Application Number | 20210260217 17/178972 |
Document ID | / |
Family ID | 1000005579724 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210260217 |
Kind Code |
A1 |
Chen; Gong ; et al. |
August 26, 2021 |
GENERATING GABAergic NEURONS IN BRAINS
Abstract
This document provides methods and materials for generating
GABAergic neurons in brains. For example, methods and materials for
using nucleic acid encoding a NeuroD1 polypeptide and nucleic acid
encoding a Dlx2 polypeptide to trigger glial cells (e.g., NG2 glial
cells or astrocytes) within the brain (e.g., striatum) into forming
GABAergic neurons (e.g., neurons resembling medium spiny neurons
such as DARPP32-positive GABAergic neurons) that are functionally
integrated into the brain of a living mammal (e.g., a human) are
provided.
Inventors: |
Chen; Gong; (State College,
PA) ; Guo; Ziyuan; (Lexington, KY) ; Wu;
Zheng; (State College, PA) ; Pei; Zifei;
(State College, PA) ; Chen; Yuchen; (Bellefonte,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Penn State Research Foundation |
University Park |
PA |
US |
|
|
Family ID: |
1000005579724 |
Appl. No.: |
17/178972 |
Filed: |
February 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15436275 |
Feb 17, 2017 |
10973930 |
|
|
17178972 |
|
|
|
|
62296960 |
Feb 18, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0085 20130101;
C12N 2740/13043 20130101; C07K 14/4702 20130101; A61K 38/1709
20130101; A61K 48/0058 20130101; A61K 48/0075 20130101; C12N 15/86
20130101; C12N 2840/007 20130101; A01K 2267/0318 20130101; A61P
25/00 20180101; A01K 2227/105 20130101; C12N 2750/14143
20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61P 25/00 20060101 A61P025/00; C07K 14/47 20060101
C07K014/47; A61K 9/00 20060101 A61K009/00; A61K 38/17 20060101
A61K038/17; C12N 15/86 20060101 C12N015/86 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
Nos. AG045656 and MH083911, awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1. (canceled)
2. A composition for converting glial cells to GABAergic neurons in
a striatum of a living mammal's brain, wherein said composition
comprises a nucleic acid vector comprising a nucleic acid sequence
encoding a neurogenic differentiation 1 (NeuroD1) polypeptide and a
nucleic acid sequence encoding a distal-less homeobox 2 (Dlx2)
polypeptide.
3. The composition of claim 2, wherein said nucleic acid vector is
a viral vector.
4. The composition of claim 3, wherein said viral vector is an
adeno-associated viral vector.
5. The composition of claim 2, wherein said nucleic acid sequence
encoding said NeuroD1 polypeptide or said nucleic acid sequence
encoding said Dlx2 polypeptide is operably linked to a promoter
sequence, wherein said promoter sequence is a constitutive promoter
sequence.
6. The composition of claim 5, wherein said constitutive promoter
sequence is selected from the group consisting of a NG2 promoter
sequence, a GFAP promoter sequence, an EF1a promoter sequence, a
CMV promoter sequence, an Aldh1L1 promoter sequence, and a CAG
promoter sequence.
7. The composition of claim 2, wherein said nucleic acid sequence
encoding said NeuroD1 polypeptide or said nucleic acid sequence
encoding said Dlx2 polypeptide is operably linked to a promoter
sequence, wherein said promoter sequence is a glial-specific
promoter sequence.
8. The composition of claim 7, wherein said glial-specific promoter
sequence is selected from the group consisting of a NG2 promoter
sequence, a GFAP promoter sequence, an A1dh1L1 promoter sequence,
and an Olig2 promoter sequence.
9. The composition of claim 2, wherein said mammal is diagnosed
with Huntington's disease.
10. The composition of claim 9, wherein said mammal is a human.
11. The composition of claim 2, wherein said glial cells are NG2
glial cells.
12. The composition of claim 2, wherein said GABAergic neurons are
DARPP32-positive.
13. The composition of claim 2, wherein said NeuroD1 polypeptide is
a human NeuroD1 polypeptide comprising an amino acid sequence of
SEQ ID NO: 1.
14. The composition of claim 2, wherein said Dlx2 polypeptide is a
human Dlx2 peptide comprising an amino acid sequence of SEQ ID NO:
2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/436,275, filed Feb. 17, 2017, which claims the benefit of
U.S. Provisional Application Ser. No. 62/296,960, filed Feb. 18,
2016. The disclosures of the prior applications are considered part
of (and are incorporated by reference in) the disclosure of this
application.
BACKGROUND
1. Technical Field
[0003] This document relates to methods and materials for
generating GABAergic neurons in brains. For example, this document
relates to methods and materials for using nucleic acid encoding a
NeuroD1 polypeptide and nucleic acid encoding a Dlx2 polypeptide to
trigger glial cells (e.g., NG2 glial cells or astrocytes) within
the brain (e.g., striatum) into forming GABAergic neurons (e.g.,
neurons resembling parvalbumin neurons or medium spiny neurons such
as DARPP32-positive GABAergic neurons) that are functionally
integrated into the brain of a living mammal (e.g., a human).
2. Background Information
[0004] Huntington's disease is mainly caused by mutations in the
gene huntingtin (HTT), resulting into the expansion of
trinucleotide CAG repeats that encode polyglutamine. When the
number of CAG repeats in a huntingtin gene exceeds 36, it will
cause disease, and the GABAergic medium spiny neurons in the
striatum are in particular vulnerable to such polyglutamine
toxicity (Ross et al., Lancet Neurol., 10:83-98 (2011); and Walker,
Lancet, 369:218-228 (2007)). Currently, there is no effective
treatment to cure Huntington's disease.
SUMMARY
[0005] This document provides methods and materials for generating
GABAergic neurons in brains. For example, this document provides
methods and materials for using nucleic acid encoding a NeuroD1
polypeptide and nucleic acid encoding a Dlx2 polypeptide to trigger
glial cells (e.g., NG2 glial cells or astrocytes) within the brain
(e.g., striatum) into forming GABAergic neurons (e.g., neurons
resembling parvalbumin neurons or medium spiny neurons such as
DARPP32-positive GABAergic neurons) that are functionally
integrated into the brain of a living mammal (e.g., a human).
[0006] As described herein, nucleic acid designed to express a
NeuroD1 polypeptide and nucleic acid designed to express a Dlx2
polypeptide can be delivered together to glial cells (e.g., NG2
glial cells or astrocytes) within a mammal's brain (e.g., striatum)
in a manner that triggers the glial cells to form functional and
integrated GABAergic neurons. These functional and integrated
GABAergic neurons can resemble medium spiny neurons (e.g., they can
be DARPP32-positive GABAergic neurons). Having the ability to form
new GABAergic neurons within the striatum of a living mammal's
brain using the methods and materials described herein can allow
clinicians and patients (e.g., Huntington's disease patients) to
create a brain architecture that more closely resembles the
architecture of a healthy brain when compared to the architecture
of an untreated Huntington's disease patient's brain following the
significant death or degeneration of GABAergic medium spiny
neurons. This can represent an important step forward for
Huntington's disease patients even though there is currently no
cure for the disease. In some cases, having the ability to
replenish GABAergic medium spiny neurons within the striatum that
die or degenerate during Huntington's disease progression using the
methods and materials described herein can allow clinicians and
patients to slow, delay, or reverse Huntington's disease
progression.
[0007] In general, one aspect of this document features a method
for forming GABAergic neurons in a striatum of a living mammal's
brain. The method comprises, or consists essentially of,
administering nucleic acid encoding a NeuroD1 polypeptide and
nucleic acid encoding a Dlx2 polypeptide (or a NeuroD1 polypeptide
and a Dlx2 polypeptide) to glial cells within the striatum, wherein
the NeuroD1 polypeptide and the Dlx2 polypeptide are expressed by
the glial cells, and wherein the glial cells form or are converted
into GABAergic neurons within the striatum. The mammal can be a
human. The glial cells can be NG2 glial cells or astrocytes. The
GABAergic neurons can be parvalbumin-positive or DARPP32-positive.
The NeuroD1 polypeptide can be a human NeuroD1 polypeptide. The
Dlx2 polypeptide can be a human Dlx2 polypeptide. The nucleic acid
encoding the NeuroD1 polypeptide can be administered to the glial
cells in the form of a viral vector. In such cases, the viral
vector can be an adeno-associated viral vector (e.g., an
adeno-associated virus serotype 2 viral vector, an adeno-associated
virus serotype 5 viral vector, or an adeno-associated virus
serotype 9 viral vector). The nucleic acid encoding the Dlx2
polypeptide can be administered to the glial cells in the form of a
viral vector. In such cases, the viral vector can be an
adeno-associated viral vector (e.g., an adeno-associated virus
serotype 2 viral vector, an adeno-associated virus serotype 5 viral
vector, or an adeno-associated virus serotype 9 viral vector). The
nucleic acid encoding the NeuroD1 polypeptide and the nucleic acid
encoding the Dlx2 polypeptide can be located on the same viral
vector, and the viral vector can be administered to the glial
cells. In such cases, the viral vector can be an adeno-associated
viral vector (e.g., an adeno-associated virus serotype 2 viral
vector, an adeno-associated virus serotype 5 viral vector, or an
adeno-associated virus serotype 9 viral vector). The nucleic acid
encoding the NeuroD1 polypeptide and the nucleic acid encoding the
Dlx2 polypeptide can be located on separate viral vectors, and each
of the separate viral vectors can be administered to the glial
cells. In such cases, each of the separate viral vectors can be an
adeno-associated viral vector (e.g., an adeno-associated virus
serotype 2 viral vector, an adeno-associated virus serotype 5 viral
vector, or an adeno-associated virus serotype 9 viral vector). The
administration can comprise a direct injection into the striatum of
the living mammal's brain. The administration can comprise an
intraperitoneal, intracranial, intravenous, intranasal, or oral
administration. The nucleic acid encoding the NeuroD1 polypeptide
can be operably linked to a promoter sequence; and the promoter
sequence can be constitutive promoter sequence. The constitutive
promoter sequence can comprise a NG2 promoter sequence, a GFAP
promoter sequence, an EF1a promoter sequence, a CMV promoter
sequence, an Aldh1L1 promoter sequence, or a CAG promoter sequence.
The nucleic acid encoding the NeuroD1 polypeptide can be operably
linked to a promoter sequence; and the promoter sequence can be a
glial-specific promoter sequence. The glial-specific promoter
sequence can comprise a NG2 promoter sequence, a GFAP promoter
sequence, an Aldh1L1 promoter sequence, or an Olig2 promoter
sequence. The nucleic acid encoding the Dlx2 polypeptide can be
operably linked to a promoter sequence; and the promoter sequence
can be constitutive promoter sequence. The constitutive promoter
sequence can comprise a NG2 promoter sequence, a GFAP promoter
sequence, an EF1a promoter sequence, a CMV promoter sequence, an
Aldh1L1 promoter sequence, or a CAG promoter sequence. The nucleic
acid encoding the Dlx2 polypeptide can be operably linked to a
promoter sequence; and the promoter sequence can be a
glial-specific promoter sequence. The glial-specific promoter
sequence can comprise a NG2 promoter sequence, a GFAP promoter
sequence, an Aldh1L1 promoter sequence, or an Olig2 promoter
sequence.
[0008] In another aspect, this document features a composition for
forming GABAergic neurons in a striatum of a living mammal's brain.
The composition comprises, or consists essentially of, a nucleic
acid vector comprising a nucleic acid sequence encoding a NeuroD1
polypeptide and a nucleic acid sequence encoding a Dlx2
polypeptide. The nucleic acid vector can be a viral vector such as
an adeno-associated viral vector (e.g., an adeno-associated virus
serotype 2 viral vector, an adeno-associated virus serotype 5 viral
vector, or an adeno-associated virus serotype 9 viral vector). The
nucleic acid sequence encoding the NeuroD1 polypeptide can be
operably linked to a promoter sequence; and the promoter sequence
can be constitutive promoter sequence. The constitutive promoter
sequence can comprise a NG2 promoter sequence, a GFAP promoter
sequence, an EF1a promoter sequence, a CMV promoter sequence, an
Aldh1L1 promoter sequence, or a CAG promoter sequence. The nucleic
acid sequence encoding the NeuroD1 polypeptide can be operably
linked to a promoter sequence; and the promoter sequence can be a
glial-specific promoter sequence. The glial-specific promoter
sequence can comprise a NG2 promoter sequence, a GFAP promoter
sequence, an Aldh1L1 promoter sequence, or an Olig2 promoter
sequence. The nucleic acid sequence encoding the Dlx2 polypeptide
can be operably linked to a promoter sequence; and the promoter
sequence can be constitutive promoter sequence. The constitutive
promoter sequence can comprise a NG2 promoter sequence, a GFAP
promoter sequence, an EF1a promoter sequence, a CMV promoter
sequence, an Aldh1L1 promoter sequence, or a CAG promoter sequence.
The nucleic acid sequence encoding the Dlx2 polypeptide can be
operably linked to a promoter sequence; and the promoter sequence
can be a glial-specific promoter sequence. The glial-specific
promoter sequence can comprise a NG2 promoter sequence, a GFAP
promoter sequence, an Aldh1L1 promoter sequence, or an Olig2
promoter sequence.
[0009] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0010] Other features and advantages of the invention will be
apparent from the following detailed description and drawings, and
from the claims.
DESCRIPTION OF DRAWINGS
[0011] FIGS. 1A-H. Conversion of cultured NG2 cells into functional
GABAergic neurons. (A) Differentiation of NG2 cells infected by
control retrovirus (expressing GFP under NG2 promoter) into
immature oligodendrocytes (CNPase-positive, red) after 3 days
post-infection (DPI). (B) NG2 cells infected by NG2::Dlx2
retrovirus were reprogrammed into neurons (NeuN-positive, red, 7
DPI). (C) NG2-converted neurons were innervated by GABAergic
synapses, as shown by GABAergic presynaptic protein GAD65 (red, 14
DPI). (D) Representative traces recorded from NG2-converted neurons
showing upward spontaneous synaptic events when holding at -20 mV
(14 DPI). Note all events were blocked by the GABA.sub.A receptor
antagonist BIC (20 .mu.M), suggesting that they were GABAergic
events. (E-F) NeuroD1 enhanced the conversion efficiency induced by
Dlx2, as shown by Tuj1 staining (E, 14 DPI) and GAD67 staining (F,
21 DPI). (G-H) Quantified data showing a significant increase of
the number of Tuj1 positive neurons (G) or GAD67 positive GABAergic
neurons (H) after coexpressing NeuroD1 with Dlx2 together. Data
were presented as mean.+-.s.e.m. ***P<0.001 (Student's t-test).
Scale bars: 40 .mu.m for panels A, B, E, and F; 10 .mu.m for panel
C.
[0012] FIGS. 2A-D. Characterizing mouse NG2 cultures. (A-C)
Infecting mouse NG2 cultures with control retrovirus NG2::GFP
revealed a small percentage of cells immunopositive for astrocyte
marker GFAP, but not microglia marker Iba1 or immature neuron
marker DCX. Scale bar: 40 .mu.m. (D) Quantified data showing the
majority of NG2 cells will differentiate into oligodendrocytes
(CNPase) (3 days after infection of NG2::GFP in differentiation
medium).
[0013] FIG. 3. No glutamatergic neurons generated from NG2 cells
after expressing Dlx2. NG2-converted neurons were immunonegative
for vGlut1, suggesting no glutamatergic neurons after reprogramming
NG2 cells by Dlx2 alone (n=60 cells in 4 repeats). Scale bar: 40
.mu.m.
[0014] FIGS. 4A-D. Screening transcriptional factors for efficient
conversion of NG2 cells into GABAergic neurons. (A) GAD67-positive
neurons converted from NG2 cells after infection with different
combinations of neural transcription factors (Dlx2, NeuroD1, and
Ascl1; 14 DPI). (B) Quantified data showing a high conversion
efficiency of NG2 cells into GABAergic neurons in Dlx2+NeuroD1 and
Dlx2+NeuroD1+Ascl1 groups. (C) Representative traces showing upward
GABAergic events recorded from NG2-converted neurons after
infection with different transcription factors (21 days post
infection). (D) Quantification of the frequency of GABAergic events
also showed a high conversion efficiency of NG2 cells into
GABAergic neurons by Dlx2+NeuroD1.
[0015] FIGS. 5A-H. Classification of NG2-converted GABAergic
neurons in culture. (A-E) Immunostaining of NG2-converted neurons
with a series of interneuron subtype markers (CR, SST, PV, CCK8 and
NPY) after infection with NeuroD1+Dlx2 retroviruses (21 DPI). Scale
bars: 40 .mu.m. (F) Quantification showing that many NG2-converted
neurons were immunopositive for calretinin (CR), somatostatin
(SST), and parvalbumin (PV), but much less for CCK8 or NPY. (G-H)
Representative traces showing different action potential firing
patterns among NG2-converted neurons (G, 12.9 Hz; H, 38.9 Hz). Note
that panel H shows an example of fast-spiking like firing
pattern.
[0016] FIGS. 6A-H. In vivo reprogramming NG2 cells into functional
GABAergic neurons. (A) Schematic diagram showing Cre-mediated FLEx
switch of the NeuroD1/Dlx2-P2A-mCherry system. (B) NG2 cells
detected after in vivo injection of NG2::Cre and FLEx-mCherry AAV
into the striatum. (C) Macroscopic view of AAV-infected striatal
region (21 DPI). (D) NeuroD1/Dlx2-infected NG2 cells showed
neuron-like morphology and NeuN staining (arrow, red) at 21 days
post AAV injection. (E) Quantified data showing a gradual decrease
of NG2 cells among infected cells, accompanied by an increase of
neurons after NeuroD1/Dlx2 infection, indicating a conversion of
NG2 cells into neuronal cells. (F-G) NG2-converted neurons (21 DPI)
in the striatum were immunopositive for GABA (F) and GAD67 (G).
Scale bars: 40 .mu.m for panel B, D; 20 .mu.m for panel F, H. (H)
Spontaneous synaptic events recorded from NG2-onverted neurons (31
DPI).
[0017] FIGS. 7A-C. Reprogramming NG2 cells into neurons under
direct control of NG2 promoter. (A) NG2 cells revealed by infection
of control virus NG2::GFP in the striatum. (B) NG2 cells became
NeuN-positive neurons after infection by NG2::NeuroD1/Dlx2. Scale
bar: 40 .mu.m. (C) Quantified data showing the percentage of
neurons versus NG2 cells after infection by NG2::GFP or
NG2::NeuroD/Dlx2. The majority of NG2 cells converted into neurons
after expressing NeuroD1+Dlx2 under the direct control of NG2
promoter.
[0018] FIGS. 8A-G. Characterizing the subtypes of NG2-converted
neurons in striatum. (A-E) Immunostaining with a series of
GABAergic neuron subtype markers (SST, PV, NPY, CCK8 and DARPP32)
in the striatum after ectopic expression of NeuroD1 and Dlx2 in NG2
cells (21 DPI). Scale bars: 40 .mu.m. (F) Quantified data showing a
significant proportion of neurons immunopositive for DARPP32 and PV
after NeuroD1/Dlx2 AAV injection into the striatum. (G)
Representative traces showing action potential firing patterns
recorded from NG2-converted neurons (31 DPI) in brain slices (n=20
neurons). Some neurons showed fast-spiking like firing pattern,
with a frequency range of 70-200 Hz.
[0019] FIGS. 9A-F. In situ reprogramming cortical NG2 cells into
functional GABAergic neurons. (A) NG2 cells revealed after
injecting NG2::Cre and FLEx-mCherry AAV into mouse prefrontal
cortex. (B) Low-magnification images showing AAV-infected site in
the prefrontal cortex (21 DPI). (C) Reprogramming cortical NG2
cells into NeuN-positive neurons after ectopic expression of
NeuroD1 and Dlx2 in NG2 cells (21 DPI). (D-E) Some NG2-converted
neurons were immunopositive for GABA (D) and GAD67 (E) in the
striatum (21 DPI). Scale bars: 40 .mu.m for panels A, C; 500 .mu.m
for panel B; 20 .mu.m for panels D, E. (F) Representative trace
showing spontaneous synaptic events recorded from in situ
NG2-converted neurons in the prefrontal cortex (35 DPI).
[0020] FIGS. 10A-G. Characterizing subtypes of NG2-converted
neurons in prefrontal cortex. (A-E) Immunostaining showing
different subtypes of GABAergic neurons among NG2-converted cells
after NeuroD1/Dlx2 infection in the prefrontal cortex (21 DPI).
Scale bars: 40 .mu.m. (F) Quantified data showing a significant
number of NG2-converted neurons in the prefrontal cortex being
immunopositive for PV and CCK8. (G) Representative traces showing
low and high frequency action potential firing patterns among
NG2-converted neurons (35 DPI) in the prefrontal cortex (n=8
neurons). Note some neuron showed fast-spiking like action
potential firing (138 Hz).
[0021] FIGS. 11A-B. Control virus infected mainly NG2 cells in the
mouse brain (A) Control AAV (NG2::Cre and FLEx-mCherry) infected
mostly NG2 cells after injection into the striatum. (B) Control AAV
(NG2::Cre and FLEx-mCherry) also infected mainly NG2 cells in the
prefrontal cortex. Scale bar: 40 .mu.m.
[0022] FIG. 12 is a listing of an amino acid sequence of a human
NeuroD1 polypeptide (SEQ ID NO:1).
[0023] FIG. 13 is a listing of an amino acid sequence of a human
Dlx2 polypeptide (SEQ ID NO:2).
[0024] FIGS. 14A-F. NeuroD1 and Dlx2 mediate glia-to-neuron
conversion in the striatum of R6/2 mice, a mouse model for
Huntington's disease. (A, B) R6/2 mice were injected with AAV5
viruses expressing NeuroD1 and Dlx2 or mCherry control in
astrocytes under the Cre-FLEx system. After 1 month, NeuroD1
(arrows) and Dlx2 (arrows) were detected in the infected cells
(arrows) in NeuroD1/Dlx2 injected mice (B), but not in the mCherry
control mice (A). (C) In the mCherry control group, the majority of
infected cells were astrocytes labeled by GFAP (arrows). (D) In the
NeuroD1/Dlx2 group, mCherry-positive cells were not co-localized
with GFAP, but some exhibited a neuron-like morphology. (E, F) The
NeuroD1/Dlx2-mediated glia-to-neuron conversion in R6/2 mouse
striatum was confirmed by NeuN staining (arrows). The Htt
aggregations in nucleus (co-localized with DAPI) were observed in
both groups, confirming they were Huntington's disease mouse model
mice.
[0025] FIGS. 15A-B. Characterization of glia-converted neurons in
the R6/2 mouse striatum. (A) NeuroD1/Dlx2-mediated glia-converted
neurons (35 DPI) in R6/2 mouse striatum were immuno-positive for
GAD67 (arrows) and GABA (arrows). (B) Some of the converted neurons
also were labeled with DARPP32 (arrows), a marker for striatal
medium spiny GABAergic neurons, demonstrating that the
glia-converted neurons can replenish the lost DARPP32 neurons in
Huntington's disease.
[0026] FIGS. 16A-D. NeuroD1 and Dlx2 expressed using AAV9 viral
vectors mediate glia-to-neuron conversion in the striatum of R6/2
mice, a mouse model for Huntington's disease. (A, B) AAV9 was
injected into the striatum, and Dlx2 and NeuroD1 were detected in
the infected cells at 10 days post injection (arrows). (C) In the
mCherry control group, the majority of infected cells were
co-localized with astrocytic marker GFAP (arrows). (D) In the
NeuroD1/Dlx2 group, most of mCherry positive cells were
co-localized with neuronal marker NeuN (arrows).
[0027] FIG. 17A-B. Characterization of glia-converted neurons in
the striatum by AAV9. (A) NeuroD1/Dlx2 converted neurons were
immuno-positive for GABAergic neuronal marker GAD67 (arrows). (B)
Some of the converted neurons also were immuno-positive for DARPP32
(arrows), a marker for striatal GABAergic medium spiny neurons.
DETAILED DESCRIPTION
[0028] This document provides methods and materials for generating
GABAergic neurons in brains. For example, this document provides
methods and materials for using nucleic acid encoding a NeuroD1
polypeptide and nucleic acid encoding a Dlx2 polypeptide to trigger
glial cells within the brain into forming GABAergic neurons that
can be functionally integrated into the brain of a living mammal.
Forming GABAergic neurons as described herein can include
converting glial cells within the brain into GABAergic neurons that
can be functionally integrated into the brain of a living mammal.
In some cases, the methods and materials described herein can be
used to improve the brain architecture of Huntington's disease
patient's brain such that it more closely resembles the brain
architecture of a healthy human, to restore a healthy brain
architecture to a Huntington's disease patient's brain, to reduce
the progression of Huntington's disease, to delay the onset of
Huntington's disease symptoms, and/or to treat Huntington's
disease. In some cases, the methods and materials described herein
can be used to reverse the effects of Huntington's disease in a
mammal with Huntington's disease.
[0029] Any appropriate mammal can be treated as described herein.
For example, mammals including, without limitation, humans,
monkeys, dogs, cats, cows, horses, pigs, rats, and mice can be
treated as described herein to generate GABAergic neurons in the
brain of a living mammal. In some cases, a human having
Huntington's disease can be treated as described herein to generate
GABAergic neurons in a Huntington's disease patient's brain. A
mammal can be identified as having Huntington's disease using any
appropriate Huntington's disease diagnostic technique. For example,
a genetic screen of the Huntingtin gene can be performed to
diagnose a human as having Huntington's disease.
[0030] As described herein, a mammal can be treated by
administering nucleic acid designed to express a NeuroD1
polypeptide and nucleic acid designed to express a Dlx2 polypeptide
to glial cells (e.g., NG2 glial cells or astrocytes) within the
mammal's brain (e.g., striatum) in a manner that triggers the glial
cells to form functional and integrated GABAergic neurons. Examples
of NeuroD1 polypeptides include, without limitation, those
polypeptides having the amino acid sequence set forth in
GenBank.RTM. accession number NP_002491 (GI number 121114306). A
NeuroD1 polypeptide can be encoded by a nucleic acid sequence as
set forth in GenBank.RTM. accession number NM_002500 (GI number
323462174). Examples of Dlx2 polypeptides include, without
limitation, those polypeptides having the amino acid sequence set
forth in GenBank.RTM. accession number NP_004396 (GI number
4758168). A Dlx2 polypeptide can be encoded by a nucleic acid
sequence as set forth in GenBank.RTM. accession number NM_004405
(GI number 84043958).
[0031] Any appropriate method can be used to deliver nucleic acid
designed to express a NeuroD1 polypeptide and nucleic acid designed
to express a Dlx2 polypeptide to glial cells within the brain of a
living mammal. For example, nucleic acid encoding a NeuroD1
polypeptide and nucleic acid encoding a Dlx2 polypeptide can be
administered to a mammal using one or more vectors such as viral
vectors. In some cases, separate vectors (e.g., one vector for
nucleic acid encoding a NeuroD1 polypeptide, and one vector for
nucleic acid encoding a Dlx2 polypeptide) can be used to deliver
the nucleic acids to glial cells. In some cases, a single vector
containing both nucleic acid encoding a NeuroD1 polypeptide and
nucleic acid encoding a Dlx2 polypeptide can be used to deliver the
nucleic acids to glial cells.
[0032] Vectors for administering nucleic acids (e.g., nucleic acid
encoding a NeuroD1 polypeptide and nucleic acid encoding a Dlx2
polypeptide) to glial cells can be prepared using standard
materials (e.g., packaging cell lines, helper viruses, and vector
constructs). See, for example, Gene Therapy Protocols (Methods in
Molecular Medicine), edited by Jeffrey R. Morgan, Humana Press,
Totowa, N.J. (2002) and Viral Vectors for Gene Therapy: Methods and
Protocols, edited by Curtis A. Machida, Humana Press, Totowa, N.J.
(2003). Virus-based nucleic acid delivery vectors are typically
derived from animal viruses, such as adenoviruses, adeno-associated
viruses, retroviruses, lentiviruses, vaccinia viruses, herpes
viruses, and papilloma viruses. In some cases, nucleic acid
encoding a NeuroD1 polypeptide and nucleic acid encoding a Dlx2
polypeptide can be delivered to glial cells using adeno-associated
virus vectors (e.g., an adeno-associated virus serotype 2 viral
vector, an adeno-associated virus serotype 5 viral vector, or an
adeno-associated virus serotype 9 viral vector), lentiviral
vectors, retroviral vectors, adenoviral vectors, herpes simplex
virus vectors, or poxvirus vector.
[0033] In addition to nucleic acid encoding a NeuroD1 polypeptide
and/or nucleic acid encoding a Dlx2 polypeptide, a viral vector can
contain regulatory elements operably linked to the nucleic acid
encoding a NeuroD1 polypeptide and/or a Dlx2 polypeptide. Such
regulatory elements can include promoter sequences, enhancer
sequences, response elements, signal peptides, internal ribosome
entry sequences, polyadenylation signals, terminators, or inducible
elements that modulate expression (e.g., transcription or
translation) of a nucleic acid. The choice of element(s) that may
be included in a viral vector depends on several factors,
including, without limitation, inducibility, targeting, and the
level of expression desired. For example, a promoter can be
included in a viral vector to facilitate transcription of a nucleic
acid encoding a NeuroD1 polypeptide and/or a Dlx2 polypeptide. A
promoter can be constitutive or inducible (e.g., in the presence of
tetracycline), and can affect the expression of a nucleic acid
encoding a polypeptide in a general or tissue-specific manner.
Examples of tissue-specific promoters that can be used to drive
expression of a NeuroD1 polypeptide and/or a Dlx2 polypeptide in
glial cells include, without limitation, NG2, GFAP, Olig2, CAG,
EF1a, Aldh1L1, and CMV promoters.
[0034] As used herein, "operably linked" refers to positioning of a
regulatory element in a vector relative to a nucleic acid in such a
way as to permit or facilitate expression of the encoded
polypeptide. For example, a viral vector can contain a
glial-specific NG2 promoter and nucleic acid encoding a NeuroD1
polypeptide or a Dlx2 polypeptide. In this case, the NG2 promoter
is operably linked to a nucleic acid encoding a NeuroD1 polypeptide
or a Dlx2 polypeptide such that it drives transcription in glial
cells.
[0035] Nucleic acid encoding a NeuroD1 polypeptide and/or a Dlx2
polypeptide also can be administered to a mammal using non-viral
vectors. Methods of using non-viral vectors for nucleic acid
delivery are described elsewhere. See, for example, Gene Therapy
Protocols (Methods in Molecular Medicine), edited by Jeffrey R.
Morgan, Humana Press, Totowa, N.J. (2002). For example, nucleic
acid encoding a NeuroD1 polypeptide and/or a Dlx2 polypeptide can
be administered to a mammal by direct injection of nucleic acid
molecules (e.g., plasmids) comprising nucleic acid encoding a
NeuroD1 polypeptide and/or a Dlx2 polypeptide, or by administering
nucleic acid molecules complexed with lipids, polymers, or
nanospheres. In some cases, a genome editing technique such as
CRISPR/Cas9-mediated gene editing can be used to activate
endogenous NeuroD1 and/or Dlx2 gene expression.
[0036] Nucleic acid encoding a NeuroD1 polypeptide and/or a Dlx2
polypeptide can be produced by techniques including, without
limitation, common molecular cloning, polymerase chain reaction
(PCR), chemical nucleic acid synthesis techniques, and combinations
of such techniques. For example, PCR or RT-PCR can be used with
oligonucleotide primers designed to amplify nucleic acid (e.g.,
genomic DNA or RNA) encoding a NeuroD1 polypeptide and/or a Dlx2
polypeptide.
[0037] In some cases, NeuroD1 polypeptides and/or Dlx2 polypeptides
can be administered in addition to or in place of nucleic acid
designed to express a NeuroD1 polypeptide and/or nucleic acid
designed to express a Dlx2 polypeptide. For example, NeuroD1
polypeptides and/or Dlx2 polypeptides can be administered to a
mammal to trigger glial cells within the brain into forming
GABAergic neurons that can be functionally integrated into the
brain of the living mammal.
[0038] Nucleic acid designed to express a NeuroD1 polypeptide and
nucleic acid designed to express a Dlx2 polypeptide (or NeuroD1
and/or Dlx2 polypeptides) can be delivered to glial cells within
the brain (e.g., glial cells within the striatum) via direct
intracranial injection, intraperitoneal administration, intranasal
administration, intravenous administration, or oral delivery in
nanoparticles and/or drug tablets, capsules, or pills.
[0039] As described herein, nucleic acid designed to express a
NeuroD1 polypeptide and nucleic acid designed to express a Dlx2
polypeptide (or NeuroD1 and/or Dlx2 polypeptides) can be
administered to a mammal (e.g., a human) having Huntington's
disease and used to improve the brain architecture of the
Huntington's disease patient's brain such that it more closely
resembles the brain architecture of a healthy human, to restore a
healthy brain architecture to a Huntington's disease patient's
brain, to reduce the progression of Huntington's disease, to delay
the onset of Huntington's disease symptoms, to treat Huntington's
disease, or to reverse the effects of Huntington's disease in the
mammal. In some cases, nucleic acid designed to express a
polypeptide having the amino acid sequence set forth in SEQ ID NO:1
and nucleic acid designed to express a polypeptide having the amino
acid sequence set forth in SEQ ID NO:2 (or a polypeptide having the
amino acid sequence set forth in SEQ ID NO:1 and/or a polypeptide
having the amino acid sequence set forth in SEQ ID NO:2) can be
administered to a mammal (e.g., a human) having Huntington's
disease as described herein and used to improve the brain
architecture of the Huntington's disease patient's brain such that
it more closely resembles the brain architecture of a healthy
human, to restore a healthy brain architecture to a Huntington's
disease patient's brain, to reduce the progression of Huntington's
disease, to delay the onset of Huntington's disease symptoms, to
treat Huntington's disease, or to reverse the effects of
Huntington's disease in the mammal. For example, a single
adeno-associated viral vector can be designed to express a
polypeptide having the amino acid sequence set forth in SEQ ID NO:1
and a polypeptide having the amino acid sequence set forth in SEQ
ID NO:2, and that designed viral vector can be administered to a
human having Huntington's disease to treat Huntington's
disease.
[0040] In some cases, a polypeptide containing the entire amino
acid sequence set forth in SEQ ID NO:1, except that the amino acid
sequence contains from one to ten (e.g., ten, one to nine, two to
nine, one to eight, two to eight, one to seven, one to six, one to
five, one to four, one to three, two, or one) amino acid additions,
deletions, substitutions, or combinations thereof, can be used. For
example, nucleic acid designed to express a polypeptide containing
the entire amino acid sequence set forth in SEQ ID NO:1 with one to
ten amino acid additions, deletions, substitutions, or combinations
thereof and nucleic acid designed to express a Dlx2 polypeptide (or
the polypeptides themselves) can be designed and administered to a
human having Huntington's disease to treat Huntington's
disease.
[0041] In some cases, a polypeptide containing the entire amino
acid sequence set forth in SEQ ID NO:2, except that the amino acid
sequence contains from one to ten (e.g., ten, one to nine, two to
nine, one to eight, two to eight, one to seven, one to six, one to
five, one to four, one to three, two, or one) amino acid additions,
deletions, substitutions, or combinations thereof, can be used. For
example, nucleic acid designed to express a polypeptide containing
the entire amino acid sequence set forth in SEQ ID NO:2 with one to
ten amino acid additions, deletions, substitutions, or combinations
thereof and nucleic acid designed to express a NeuroD1 polypeptide
(or the polypeptides themselves) can be designed and administered
to a human having Huntington's disease to treat Huntington's
disease. In another example, nucleic acid designed to express a
polypeptide containing the entire amino acid sequence set forth in
SEQ ID NO:1 with one to ten amino acid additions, deletions,
substitutions, or combinations thereof and nucleic acid designed to
express a polypeptide containing the entire amino acid sequence set
forth in SEQ ID NO:2 with one to ten amino acid additions,
deletions, substitutions, or combinations thereof can be designed
and administered to a human having Huntington's disease to treat
Huntington's disease.
[0042] Any appropriate amino acid residue set forth in SEQ ID NO:1
and/or SEQ ID NO:2 can be deleted, and any appropriate amino acid
residue (e.g., any of the 20 conventional amino acid residues or
any other type of amino acid such as ornithine or citrulline) can
be added to or substituted within the sequence set forth in SEQ ID
NO:1 and/or SEQ ID NO:2. The majority of naturally occurring amino
acids are L-amino acids, and naturally occurring polypeptides are
largely comprised of L-amino acids. D-amino acids are the
enantiomers of L-amino acids. In some cases, a polypeptide as
provided herein can contain one or more D-amino acids. In some
embodiments, a polypeptide can contain chemical structures such as
.epsilon.-aminohexanoic acid; hydroxylated amino acids such as
3-hydroxyproline, 4-hydroxyproline, (5R)-5-hydroxy-L-lysine,
allo-hydroxylysine, and 5-hydroxy-L-norvaline; or glycosylated
amino acids such as amino acids containing monosaccharides (e.g.,
D-glucose, D-galactose, D-mannose, D-glucosamine, and
D-galactosamine) or combinations of monosaccharides.
[0043] Amino acid substitutions can be made, in some cases, by
selecting substitutions that do not differ significantly in their
effect on maintaining (a) the structure of the peptide backbone in
the area of the substitution, (b) the charge or hydrophobicity of
the molecule at particular sites, or (c) the bulk of the side
chain. For example, naturally occurring residues can be divided
into groups based on side-chain properties: (1) hydrophobic amino
acids (norleucine, methionine, alanine, valine, leucine, and
isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine,
and threonine); (3) acidic amino acids (aspartic acid and glutamic
acid); (4) basic amino acids (asparagine, glutamine, histidine,
lysine, and arginine); (5) amino acids that influence chain
orientation (glycine and proline); and (6) aromatic amino acids
(tryptophan, tyrosine, and phenylalanine). Substitutions made
within these groups can be considered conservative substitutions.
Non-limiting examples of substitutions that can be used herein for
SEQ ID NO:1 and/or SEQ ID NO:2 include, without limitation,
substitution of valine for alanine, lysine for arginine, glutamine
for asparagine, glutamic acid for aspartic acid, serine for
cysteine, asparagine for glutamine, aspartic acid for glutamic
acid, proline for glycine, arginine for histidine, leucine for
isoleucine, isoleucine for leucine, arginine for lysine, leucine
for methionine, leucine for phenyalanine, glycine for proline,
threonine for serine, serine for threonine, tyrosine for
tryptophan, phenylalanine for tyrosine, and/or leucine for valine.
Further examples of conservative substitutions that can be made at
any appropriate position within SEQ ID NO:1 and/or SEQ ID NO:2 are
set forth in Table 1.
TABLE-US-00001 TABLE 1 Examples of conservative amino acid
substitutions. Original Exemplary Preferred Residue substitutions
substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln,
His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn Asn Glu Asp Asp
Gly Pro Pro His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe,
Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg,
Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala Leu Pro
Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser
Phe Val Ile, Leu, Met, Phe, Ala, Norleucine Leu
[0044] In some embodiments, polypeptides can be designed to include
the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2
with the proviso that it includes one or more non-conservative
substitutions. Non-conservative substitutions typically entail
exchanging a member of one of the classes described above for a
member of another class. Whether an amino acid change results in a
functional polypeptide can be determined by assaying the specific
activity of the polypeptide using, for example, the methods
disclosed herein.
[0045] In some cases, a polypeptide having an amino acid sequence
with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99.0%) sequence identity to the
amino acid sequence set forth in SEQ ID NO:1, provided that it
includes at least one difference (e.g., at least one amino acid
addition, deletion, or substitution) with respect to SEQ ID NO:1,
can be used. For example, nucleic acid designed to express a
polypeptide containing an amino acid sequence with between 90% and
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:1 and nucleic acid designed to express a Dlx2 polypeptide (or
the polypeptides themselves) can be designed and administered to a
human having Huntington's disease to treat Huntington's
disease.
[0046] In some cases, a polypeptide having an amino acid sequence
with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99.0%) sequence identity to the
amino acid sequence set forth in SEQ ID NO:2, provided that it
includes at least one difference (e.g., at least one amino acid
addition, deletion, or substitution) with respect to SEQ ID NO:2,
can be used. For example, nucleic acid designed to express a
polypeptide containing an amino acid sequence with between 90% and
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:2 and nucleic acid designed to express a NeuroD1 polypeptide
(or the polypeptides themselves) can be designed and administered
to a human having Huntington's disease to treat Huntington's
disease. In another example, nucleic acid designed to express a
polypeptide containing an amino acid sequence with between 90% and
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:1 and nucleic acid designed to express a polypeptide
containing an amino acid sequence with between 90% and 99% sequence
identity to the amino acid sequence set forth in SEQ ID NO:2 (or
the polypeptides themselves) can be designed and administered to a
human having Huntington's disease to treat Huntington's
disease.
[0047] Percent sequence identity is calculated by determining the
number of matched positions in aligned amino acid sequences,
dividing the number of matched positions by the total number of
aligned amino acids, and multiplying by 100. A matched position
refers to a position in which identical amino acids occur at the
same position in aligned amino acid sequences. Percent sequence
identity also can be determined for any nucleic acid sequence.
[0048] The percent sequence identity between a particular nucleic
acid or amino acid sequence and a sequence referenced by a
particular sequence identification number (e.g., SEQ ID NO:1 or SEQ
ID NO:2) is determined as follows. First, a nucleic acid or amino
acid sequence is compared to the sequence set forth in a particular
sequence identification number using the BLAST 2 Sequences (Bl2seq)
program from the stand-alone version of BLASTZ containing BLASTN
version 2.0.14 and BLASTP version 2.0.14. This stand-alone version
of BLASTZ can be obtained online at fr.com/blast or at
ncbi.nlm.nih.gov. Instructions explaining how to use the Bl2seq
program can be found in the readme file accompanying BLASTZ. Bl2seq
performs a comparison between two sequences using either the BLASTN
or BLASTP algorithm. BLASTN is used to compare nucleic acid
sequences, while BLASTP is used to compare amino acid sequences. To
compare two nucleic acid sequences, the options are set as follows:
-i is set to a file containing the first nucleic acid sequence to
be compared (e.g., C:\seq1.txt); -j is set to a file containing the
second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p
is set to blastn; -o is set to any desired file name (e.g.,
C:\output.txt); -q is set to -l; -r is set to 2; and all other
options are left at their default setting. For example, the
following command can be used to generate an output file containing
a comparison between two sequences: C:\Bl2seq c:\seq1.txt -j
c:\seq2.txt -p blastn -o c:\output.txt -q -l -r 2. To compare two
amino acid sequences, the options of Bl2seq are set as follows: -i
is set to a file containing the first amino acid sequence to be
compared (e.g., C:\seq1.txt); -j is set to a file containing the
second amino acid sequence to be compared (e.g., C:\seq2.txt); -p
is set to blastp; -o is set to any desired file name (e.g.,
C:\output.txt); and all other options are left at their default
setting. For example, the following command can be used to generate
an output file containing a comparison between two amino acid
sequences: C:\Bl2seq c:\seq1.txt -j c:\seq2.txt -p blastp -o
c:\output.txt. If the two compared sequences share homology, then
the designated output file will present those regions of homology
as aligned sequences. If the two compared sequences do not share
homology, then the designated output file will not present aligned
sequences.
[0049] Once aligned, the number of matches is determined by
counting the number of positions where an identical nucleotide or
amino acid residue is presented in both sequences. The percent
sequence identity is determined by dividing the number of matches
by the length of the sequence set forth in the identified sequence
(e.g., SEQ ID NO:1), followed by multiplying the resulting value by
100. For example, an amino acid sequence that has 340 matches when
aligned with the sequence set forth in SEQ ID NO:1 is 95.5 percent
identical to the sequence set forth in SEQ ID NO:1 (i.e.,
340.+-.356.times.100=95.5056). It is noted that the percent
sequence identity value is rounded to the nearest tenth. For
example, 75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1,
while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2.
It also is noted that the length value will always be an
integer.
[0050] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1--In Vivo Reprogramming Glial Cells into GABAergic Neurons
in the Striatum to Treat Huntington's Disease
NG2 Cell Culture
[0051] As described elsewhere (Guo et al., Cell Stem Cell,
14:188-202 (2014)), mouse cortical tissue was dissected out and
isolated from the brain of postnatal pups (P3-P5). Cortical cells
were dissociated (0.25% trypsin-EDTA) and plated in 25 cm.sup.2
flasks coated with poly-D-lysine (PDL, Sigma), and cultured in
DMEM/F12 (GIBCO) with 10% fetal bovine serum (FBS, GIBCO) for 9
days, with the medium changed once every 3 days. On the ninth day,
the flasks were rigorously shook, and the supernatant was collected
and centrifuged to harvest NG2 cells with a small number of
neurons, astrocytes, and microglial cells. The majority of
astrocytes were flat and not easy to shake off the flasks. After
centrifuge, cells were resuspended and seeded on PDL-coated
coverslips (12 mm). The NG2 cells were maintained in serum-free
DMEM medium (GIBCO) supplied with N2 supplements (STEMCELL), 10
ng/mL platelet-derived growth factor (PDGF, Invitrogen), 10 ng/mL
FGF2 (Invitrogen), and 10 ng/mL EGF (Invitrogen), at 37.degree. C.
in humidified air with 5% CO2.
Retrovirus Production
[0052] The human NG2 promoter gene was subcloned from hNG2
Promoter-GLuc (GeneCopoeia) and used to replace the CAG promoter in
pCAG retroviral vector, which encoded either NeuroD1 or GFP, as
described elsewhere (Guo et al., Cell Stem Cell, 14:188-202
(2014)), to generate pNG2-NeuroD1-IRES-GFP or pNG2-GFP-IRES-GFP.
The mouse Dlx2 cDNA was subcloned from pCAG-Dlx2-IRES-DsRed
(Heinrich et al., PloS Biology, (2010)) (obtained from Dr.
Magdalena Gotz) and inserted into pNG2 retroviral vector to
generate pNG2-Dlx2-IRES-GFP. The E2A-Dlx2 cDNA was a PCR product
from the template plasmid pCAG-Dlx2-IRES-DsRed using a 5' primer
containing an E2A peptide. This PCR product was inserted into pNG2
retroviral vector to generate pNG2-NeuroD1-E2A-Dlx2-IRES-GFP. The
pCAG-NeuroD1-IRES-GFP was constructed as described elsewhere (Guo
et al., Cell Stem Cell, 14:188-202 (2014)). The human ASCL1 plasmid
was constructed from a PCR product using a template of the
pCMV6-XL5-ASCL1 (OriGene) that was inserted into a
pCAG-GFP-IRES-GFP retroviral vector (Zhao et al., J. Neurosci.,
(2006)) (obtained from Dr. Fred Gage) to generate
pCAG-ASCL1-IRES-GFP. To package retroviral particles, the target
plasmid described above were transfected into gpg helper-free human
embryonic kidney (HEK) cells to generate vesicular stomatitis virus
glycoprotein (VSV-G)-pseudotyped retroviruses encoding neural
transcription factors. The titer of viral particles was about
10.sup.7 particles/mL, determined after transduction of HEK
cells.
Trans-Differentiation of NG2 Cells into Neurons
[0053] Twenty-four hours after infection of mouse NG2 cells with
retroviruses, the culture medium was replaced by a differentiation
medium that included DMEM/F12 (GIBCO), 0.5% FBS (GIBCO), N2
supplements (GIBCO), vitamin C (VC, 5 .mu.g/mL, Sigma), ROCK
inhibitor (Y-27632, 1 .mu.M, Selleckchem), and
penicillin/streptomycin (GIBCO). To promote synaptic maturation
during conversion, brain-derived neurotrophic factor (BDNF, 20
ng/mL, Invitrogen) was added to the cultures every four days. Due
to the morphological change from NG2 cells to neurons during
reprogramming, the empty space was filled with additional mouse
astrocytes to support the functional development of converted
neurons.
AAV Production
[0054] The hNG2 or hGFAP promoter was amplified by PCR and inserted
into pAAV-MCS (Cell Biolab) between MluI and SacII to replace the
CMV promoter. The Cre gene was obtained by PCR from hGFAP
Promoter-Cre-MP-1 (Addgene) and inserted into pAAV-MCS EcoRI and
SalI sites to generate pAAV-NG2::Cre and pAAV-GFAP::Cre. The Cre
gene was subcloned into pAAV-MCS EcoRI and SalI sites to generate
pAAV-NG2::Cre and pAAV-GFAP::Cre. To construct
pAAV-FLEX-mCherry-P2A-mCherry (or pAAV-FLEX-GFP-P2A-GFP),
pAAV-FLEX-NeuroD1-P2A-mCherry (or pAAV-FLEX-NeuroD1-P2A-GFP), and
pAAV-FLEX-Dlx2-P2A-mCherry, the cDNAs coding NeuroD1, Dlx2,
mCherry, or GFP were obtained by PCR using the retroviral
constructs. The amplicons were fused with P2A-mCherry or P2A-GFP
and subcloned into the pAAV-FLEX-GFP KpnI and XhoI sites. To
generate pAAV-NG2::NeuroD1-P2A-GFP and pAAV-NG2::Dlx2-P2A-GFP, the
proneural genes, NeuroD1 or Dlx2 fused with P2A-GFP, were subcloned
into EcoRI and SalI sites after hNG2 promoter. Sequencing of the
plasmid constructs was carried out to verify their identities.
AAV5 Production and Purification
[0055] Recombinant AAV5 was produced in 293AAV cells (Cell
Biolabs). Briefly, polyethylenimine (PEI, linear, MW 25,000) was
used for transfection of triple plasmids: the pAAV expression
vector, pAAV5-RC (Cell Biolab) and pHelper (Cell Biolab). 72 hours
post transfection, cells were scrapped in their medium and
centrifuged, and frozen and thawed four times by placing it
alternately in dry ice/ethanol and 37.degree. C. water bath. AAV
crude lysate was purified by centrifugation at 54,000 rpm for 1
hour in discontinuous iodixanol gradients with a Beckman SW55Ti
rotor. The virus-containing layer was extracted, and viruses were
concentrated by Millipore Amicon Ultra Centrifugal Filters. Virus
titers were 1.2.times.10.sup.12 GC/mL for GFAP::Cre and NG2::Cre,
and 1.4.times.10.sup.12 GC/mL for FLEx-NeuroD1-P2A-GFP,
FLEx-NeuroD1-P2A-mCherry and FLEx-Dlx2-P2A-mCherry, and
1.6.times.10.sup.12 GC/mL for FLEx-mCherry-P2A-mCherry,
FLEx-GFP-P2A-GFP, NG2::NeuroD1-P2A-GFP and NG2::Dlx2-P2A-GFP were
determined by QuickTiter.TM. AAV Quantitation Kit (Cell
Biolabs).
Animals
[0056] All animals (C57/BL6 mice, 2-3 month old) were housed in a
12-hour light/dark cycle and fed with enough food and water.
Stereotaxic Viral Injection
[0057] Brain surgeries were conducted on 2-3 month-old C57/BL6 mice
for AAV injection. The mice were anesthetized by injecting 20 mL/kg
2.5% Avertin (a mixture of 25 mg/mL of Tribromoethylethanol and 25
.mu.L/ml T-amylalcohol) or ketamine/xylazine (120 mg/kg and 16
mg/kg) into the peritoneum and then placed in a stereotaxic setup.
Artificial eye ointment was applied to cover the eye for protection
purpose. The mice were operated with a midline scalp incision and
were drilled with a hole (.about.1 mm) on the skull for needle
injection, with its position relative to Bregma as following: AP
+0.6 mm, ML 1.7 mm, DV -2.8 mm for striatum; AP +1.8 mm, ML 2.5 mm,
DV -1.8 mm for prefrontal cortex. Each mouse received an injection
of AAV using a 5 .mu.L syringe and a 34G needle. The injection
volume was 2 .mu.L, and the flow rate was controlled at 0.2
.mu.L/minute. After viral injection, the needle was kept in place
for at least five additional minutes before slowly withdrawn.
Immunocytochemistry
[0058] For brain slice immunostaining, the animals were
anesthetized with 2.5% Avertin and then quickly perfused with
saline (0.9% NaCl) to wash away the blood followed with 4%
paraformaldehyde (PFA) to fix the brains. The brains were then
taken out and postfixed in 4% PFA overnight in cold room. After
fixation, the samples were cut at 35 .mu.m coronal sections by a
vibratome (Leica), washed three times by PBS, and permeabilized in
0.3% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) for
one hour. For GABA staining, the permeabilized step was skipped.
All brain sections were blocked in blocking buffer (2.5% normal
goat serum (NGS), 2.5% normal donkey serum (NDS), and 0.1% Triton
X-100 in PBS) for another hour. For cell culture immunostaining,
cells were fixed in 4% PFA in PBS for 12 minutes at room
temperature. After fixation, the cultures were first washed three
times by PBS and then permeabilized in 0.1% Triton X-100 in PBS for
30 minutes. All samples were blocked by blocking buffer for one
hour before incubation with primary antibodies.
[0059] Primary antibodies, dissolved in blocking buffer, were
incubated either with brain sections or culture samples overnight
in cold room. After washing three times in PBS, the samples were
incubated with appropriate secondary antibodies conjugated to
DyLight 488, DyLight 594, Alexa Flour 647 and Cy3 (1:1000, Jackson
ImmunoResearch) for one hour at room temperature, followed by
extensive washing in PBS. Coverslips were finally mounted onto a
glass slide with an anti-fading mounting solution with DAPI
(Invitrogen). All images were taken by a confocal microscope
(Olympus FV1000). Z-stacks of confocal images were acquired and
analyzed using FV10-ASW 3.0 Viewer software (Olympus).
Antibodies
[0060] The following primary antibodies were used: polyclonal
anti-green fluorescent protein (GFP, chicken, 1:2000, Abcam,
AB13970), polyclonal anti-glial fibrillary acidic protein (GFAP,
rabbit, 1:1000, Dako, Z0334), monoclonal anti-CNPase (mouse, 1:800,
Abcam, AB6319), polyclonal anti-vesicular glutamate transporter 1
(vGluT1, rabbit, 1:1000, Synaptic Systems), monoclonal anti GAD67
(mouse, 1:1000, Millipore, MAB5406), monoclonal anti GAD65 (GAD-6,
mouse, 1:1000, Developmental Studies Hybridoma Bank, Iowa City),
monoclonal anti-NG2 (mouse, 1:600, Abcam, AB50009), polyclonal
anti-Iba1 (rabbit, 1:1000, Wako, 019-19741), polyclonal anti-NeuN
(rabbit, 1:1000, Millipore, ABN78), monoclonal anti-.beta.III
tubulin (Tuj1, mouse, 1:1000, COVANCE, MMS-435P), polyclonal
anti-.gamma.-aminobutyric acid (GABA, rabbit, 1:2000, Sigma,
A2052), polyclonal anti-Red Fluorescent Protein (RFP, rat, 1:1500,
antibody-online, ABIN334653; and rabbit, 1:1000, Rockland,
600-401-379), polyclonal anti-T-box brain 1 (Tbr1, 1:800, rabbit,
Abcam, AB31940), monoclonal anti-Ctip2 (Rat, 1:1000, Abcam,
AB18465), monoclonal anti-NeuroD1 (mouse, 1:1000, Abcam, AB60704),
polyclonal anti-Dlx2 (rabbit, 1:600, Abcam, AB30339), polyclonal
anti-Doublecortin (DCX, rabbit, 1:1000, Abcam, AB18723), monoclonal
anti-Parvalbumin (PV, mouse, 1:1000, sigma, P3088), polyclonal
anti-Calretinin (CR, goat, 1:1500, Millipore, AB1550), monoclonal
anti-Somatostatin (SST, rat, 1:300, Millipore, MAB354), polyclonal
anti-Cholecystokinin (Catalog Nos. 26-33) (CCK-8, rabbit, 1:2000,
sigma, C2581), polyclonal anti-Neuropeptide Y (NPY, rabbit, 1:2000,
Abcam, AB30914), and polyclonal anti-Dopamine- and cAMP-regulated
neuronal phosphoprotein (DARP-32, rabbit, 1:1500, Millipore,
AB10518).
Image Analysis
[0061] Cell counts were performed by taking images of several
randomly chosen views per coverslip or brain slice and analyzed by
Image J software. Data were presented as mean.+-.SEM. Student's
t-test (paired or unpaired) was used for statistical analysis.
*P<0.05, **P<0.01, ***P<0.001.
Electrophysiology
Patch-Clamp Recordings in Cell Cultures
[0062] Whole-cell recordings were conducted using Multiclamp 700A
patch-clamp amplifier (Molecular Devices, Palo Alto, Calif.) as
described elsewhere (Guo et al., Cell Stem Cell, 14:188-202
(2014)). The recording chamber was continuously perfused with a
bath solution consisting of 128 mM NaCl, 30 mM glucose, 25 mM
HEPES, 5 mM KCl, 1 mM MgCl.sub.2, and 2 mM CaCl.sub.2. The bath
solution, with an osmolarity at 315-325 mOsm/L, was adjusted to pH
7.3 by NaOH. Patch pipettes were pulled from borosilicate glass
(3-5 M.OMEGA.) and filled with an internal solution consisting of
125 mM KGluconate, 10 mM KCl, 10 mM Tris-phosphocreatine, 10 mM
HEPES, 5 mM EGTA, 4 mM MgATP, and 0.5 mM Na.sub.2GTP (pH 7.3,
adjusted with KOH) for recording action potentials. A different
internal solution composed of 135 mM CsGluconate, 5 mM EGTA, 10 mM
HEPES, 10 mM Tris-phosphocreatine, 4 mM MgATP, and 0.5 mM
Na.sub.2GTP (pH 7.3, adjusted with KOH) was used for recording
GABAergic synaptic responses (IPSCs). The series resistance was
typically 15-30 M.OMEGA. and not compensated to avoid increased
noise associated with compensation. GABA.sub.A receptor antagonist
bicuculline (Bic) was applied through a gravity-driven drug
delivery system (VC-6, Warner Hamden, Conn.). For voltage-clamp
experiments, the membrane potential was typically held at -20 or 0
mV for recording upward IPSCs. Data were acquired using pClamp 9
software (Molecular Devices, Palo Alto, Calif.), sampled at 10 kHz,
and filtered at 1 kHz. Action potentials were analyzed using pClamp
9 Clampfit software, and spontaneous synaptic events were analyzed
using MiniAnalysis software (Synaptosoft, Decator, Ga.). All
experiments were performed at room temperature.
[0063] Brain slice recordings Brain slices were prepared at 1-1.5
month after AAV injection, and cut at 300 .mu.m thick coronal
sections with a Leica vibratome in ice-cold cutting solution (75 mM
sucrose, 87 mM NaCl, 0.5 mM CaCl.sub.2, 2.5 mM KCl, 7 mM
MgCl.sub.2, 1.25 mM NaH.sub.2PO.sub.4, 25 mM NaHCO.sub.3, and 20 mM
glucose). Slices were incubated in artificial cerebral spinal fluid
(ACSF) containing: 119 mM NaCl, 2.5 mM KCl, 26 mM NaHCO.sub.3, 1.25
mM NaH.sub.2PO.sub.4, 2.5 mM CaCl.sub.2, 1.3 mM MgCl.sub.2, and 10
mM glucose, and constantly bubbled with 95% O.sub.2 and 5% CO2 at
32-33.degree. C. for 30 minutes. Brain slices were then transferred
to a chamber at room temperature to recover for one hour. The
recording chamber was set at 32-33.degree. C. for all recordings.
Whole-cell recordings were conducted using a pipette solution
consisting of 135 mM K-Gluconate, 5 mM Na-phosphocreatine, 10 mM
KCl, 2 mM EGTA, 10 mM HEPES, 4 mM MgATP, and 0.5 mM Na.sub.2GTP (pH
7.3, adjusted with KOH, 290 mOsm/L). Pipette resistance was
typically 4-6 M.OMEGA., and series resistance was around 20-40
M.OMEGA.. The membrane potential was held at -70 mV when recording
spontaneous events. Data were collected using pClamp 9 software
(Molecular Devices, Palo Alto, Calif.), sampled at 10 kHz, and
filtered at 1 kHz, then analyzed with pClamp 9 Clampfit and
MiniAnalysis software (Synaptosoft, Decator, Ga.).
Reprogramming Cultured NG2 Cells into Functional GABAergic
Neurons
[0064] NG2 cells are glial progenitor cells that mainly produce
oligodendrocytes in both physiological and pathological conditions
(Kang et al., Neuron, 68:668-681 (2010); Buffo et al., Proc. Natl.
Acad. Sci. USA, 105:3581-3586 (2008); and Nishiyama et al., Nat.
Rev. Neurosci., 10:9-22 (2009)). The following was performed to
test whether Dlx2, a transcription factor critical for GABAergic
fate determination, can reprogram NG2 cells into GABAergic
neurons.
[0065] First, primary culture of mouse NG2 cells were used to test
Dlx2 reprogramming capability. The cell cultures were enriched with
NG2 cells, as demonstrated by successful infection with retrovirus
expressing GFP under the control of human NG2 promoter
(NG2::GFP-IRES-GFP). The majority of cells were immunopositive for
oligodendrocyte marker CNPase (FIGS. 1A and 2D; 71.5.+-.5.8%
CNPase+; n=4 batches). In contrast, only about 10% of cells were
GFAP-positive astrocytes, and none were NeuN-positive neurons (FIG.
2). The NG2 cultures were infected with retrovirus expressing Dlx2
under the control of NG2 promoter (NG2::Dlx2-IRES-GFP). Many
Dlx2-infected NG2 cells became immunopositive for NeuN, a neuronal
marker, after one week of infection (FIG. 1B; 57.1.+-.5.1% NeuN+;
n=5 batches; 7 DPI), suggesting that Dlx2 is capable of
reprogramming NG2 cells into neuronal cells. Importantly, these
NG2-reprogrammed neurons induced by Dlx2 exhibited many GABAergic
synapses (FIG. 1C), but few glutamatergic synapses (FIG. 3),
suggesting that NG2 cells are likely converted into GABAergic
neurons but not glutamatergic neurons. To corroborate the
immunostaining results, the functional properties of NG2-converted
neurons were examined by patch-clamp recordings after two weeks of
Dlx2 infection. Significant GABAergic synaptic events were detected
in NG2-converted neurons (FIG. 1D; IPSC frequency, 0.7.+-.0.2 Hz;
amplitude, 19.6.+-.3.7 pA; n=7; 14 DPI), which were mostly blocked
by GABA.sub.A receptor antagonist bicuculline (BIC, 20 .mu.M),
confirming that NG2-converted neurons are GABAergic. Thus, Dlx2 can
reprogram cultured NG2 cells into functional GABAergic neurons.
Co-Expression of NeuroD1 and Dlx2 Increases the Conversion
Efficiency
[0066] While Dlx2 alone was capable of reprogramming NG2 cells into
GABAergic neurons, the reprogramming efficiency was not very high.
To increase the reprogramming efficiency, other neural
transcription factors such as Ascl1 (also known as Mash1;
Vierbuchen et al., Nature, 463:1035-1041 (2010) and Bertrand et
al., Nat. Rev. Neurosci., 3:517-530 (2002)) and NeuroD1 (Guo et
al., Cell Stem Cell, 14:188-202 (2014) and Kuwabara et al., Nat.
Neurosci., 12:1097-1105 (2009)), as well as combinations of Dlx2
with Ascl1 and NeuroD1, were tested (FIG. 4). Among all the
combinations tested, co-overexpression of NeuroD1 and Dlx2 together
generated the most number of GABAergic neurons (FIG. 4A-B, n=3
repeats, 14 DPI). Functional assay were used with
electrophysiological recordings to analyze GABAergic events among
different groups (FIG. 4C). The highest frequency of GABAergic
events was also detected in the NeuroD1+Dlx2 group (FIG. 3D, n=3
repeats, 21 DPI), consistent with the immunostaining results.
[0067] A polycistronic retroviral vector expressing NeuroD1 and
Dlx2 together under the control of NG2 promoter
(NG2::NeuroD1-E2A-Dlx2-IRES-GFP) was constructed. The new
retrovirus simultaneously expressing NeuroD1 and Dlx2 generated
many more neurons than Dlx2 alone (FIG. 1E). Quantitatively, the
Tuj1.sup.+ neurons in the NeuroD1+Dlx2 group (8.2.+-.1.0 cells/0.1
mm.sup.2) were 3-fold more than that infected by Dlx2 alone
(2.3.+-.0.3 cells/0.1 mm.sup.2; n=4 repeats; 12-14 DPI) (FIG. 1G).
The conversion efficiency also increased from 57.2.+-.8.8% by Dlx2
alone to 94.9.+-.2.1% by NeuroD1+Dlx2 together. GAD67 staining was
performed, which confirmed that the NeuroD1+Dlx2 converted neurons
were mainly GABAergic (FIGS. 1F and 1H; NeuroD1+Dlx2, 8.8.+-.0.9
cells/0.1 mm.sup.2; Dlx2, 1.4.+-.0.2 cells/0.1 mm.sup.2; n=4
repeats; 19-21 DPI). Glutamatergic neurons were <3% after
NeuroD1+Dlx2 conversion as revealed by VGlut1 staining
(2.5.+-.1.2%; n=4 repeats; 19-21 DPI). Therefore, NeuroD1
significantly facilitates Dlx2-mediated reprogramming of NG2 cells
into functional GABAergic neurons.
NG2-Converted GABAergic Neurons have Specific Subtype
Properties
[0068] GABAergic interneurons have been classified into many
subtypes according to specific protein expression markers, such as
but not limited to calretinin (CR), parvalbumin (PV), somatostatin
(SST), neuropeptide Y (NPY), and cholecystokinin (CCK) (Kepecs
& Fishell, Nature, 505:318-326 (2014); Kawaguchi & Kondo,
J. Neurocytol., 31:277-287 (2002); and Nat. Rev. Neurosci.,
9:557-568 (2008)). Therefore, a series of immunostaining was
performed with a variety of GABAergic markers to determine what
specific subtypes of GABAergic neurons were converted from NG2
cells (FIG. 5). Interestingly, nearly 90% of NG2-converted neurons
were immunopositive for CR or SST (FIG. 5A-B), over 50% were
PV-positive (FIG. 5C), and less cells positive for CCK or NPY (FIG.
5D-E). Quantitative results were shown in FIG. 5F (CR,
91.8.+-.5.6%; SST, 89.3.+-.5.5%; PV, 53.9.+-.7.8%; CCK8,
23.3.+-.9.9%; NPY, 14.9.+-.3.9%; n=3 batches; 19-21 DPI). GABAergic
neurons also can be characterized according to their firing
patterns, such as fast-spiking action potentials found in
parvalbumin neurons (Markram et al., Nat. Rev. Neurosci., 5:793-807
(2004)). When analyzed the NG2-converted neurons with patch-clamp
recordings, after NeuroD1-Dlx2 retroviral infection, some neurons
fired fast-spiking like action potentials whereas others fired
lower frequency action potentials (FIG. 5G-H, n=15 cells),
supporting that NG2-converted neurons are a mixture of PV and
non-PV interneurons. These results demonstrate that Dlx2 together
with NeuroD1 can efficiently convert cultured NG2 cells into mature
GABAergic neurons with a variety of subtypes, including PV, CR, and
SST neurons.
In Vivo Reprogramming of NG2 Cells into Functional GABAergic
Neurons
[0069] After reprogramming cultured NG2 cells into GABAergic
neurons in vitro, the following was performed to examine whether
NG2 cells in the mouse brain in vivo also can be converted into
GABAergic interneurons. As demonstrated elsewhere (Guo et al., Cell
Stem Cell, 14:188-202 (2014)), the in vivo reprogramming efficiency
induced by retroviruses is not very high. In an attempt to overcome
this, recombinant adeno-associated virus (serotype 5, rAAV5) were
made for in vivo reprogramming. Among different serotypes of rAAV,
rAAV5 was chosen for the majority of the experiments because it
prefers to infect glial cells over neuronal cells (Howard et al.,
Virology, 372:24-34 (2008) and Markakis et al., Mol. Ther.,
18:588-593 (2010)). In order to specifically target NG2 cells and
achieve stable transgene insertion, a Cre-FLEx (flip-excision)
system, which includes a vector expressing Cre recombinase under
the control of human NG2 promoter (NG2::Cre) and a vector with
Cre-mediated FLEx switch of the inverted coding sequence of
NeuroD1-P2A-mCherry or Dlx2-P2A-mCherry (FIG. 6A; Schnutgen et al.,
Nat. Biotechnol., 21:562-565 (2003) and Atasoy et al., J.
Neurosci., 28:7025-7030 (2008)), was developed. The Cre-FLEx rAAV
system was designed to allow Cre expression in NG2 cells, which in
turn will activate the transcription of NeuroD1 or Dlx2 together
with mCherry. For a control experiment, rAAV-NG2::Cre together with
rAAV-FLEx-mCherry were first injected into the mouse striatum, a
brain region enriched with GABAergic interneurons. Control rAAV
(NG2::Cre and FLEx-mCherry) successfully infected NG2 cells in the
striatum as revealed by NG2 immunostaining (FIG. 6B; 66.7.+-.11.1%
infected cells were NG2+; n=3 animals). Interestingly, after
injecting rAAV-NG2::Cre plus rAAV-FLEx-NeuroD1 and rAAV-FLEx-Dlx2
into the striatum (FIG. 6C, Cre+NeuroD1+Dlx2), many infected cells
were observed with neuron-like morphology and immunopositive for
NeuN (FIG. 6D; 80.8.+-.1.9% mCherry-labeled cells were also NeuN
positive, n=4 animals; 21 DPI). To investigate the time course of
NG2-neuron conversion, the percentage of NG2 cells versus neurons
among the total number of viral infected cells (mCherry positive)
from 4 to 21 days post viral injection (Cre+NeuroD1+Dlx2) was
examined. Interestingly, a steady decrease of NG2 cells accompanied
with a steady increase of Tuj1.sup.+ or NeuN.sup.+ neurons was
observed during this conversion period (FIG. 6E), suggesting that
NG2 cells are gradually converted into neurons.
[0070] To further demonstrate direct conversion of NG2 glia into
neurons, AAV5 vectors were constructed using NG2 promoter to
directly drive the expression of NeuroD1 or Dlx2
(NG2::NeuroD1-P2A-GFP+NG2::Dlx2-P2A-GFP), without using Cre/FLEx
system. Compared to GFP expression alone under the control of NG2
promoter (NG2::GFP), many more neurons (NeuN-positive) were
detected after expressing NeuroD1+Dlx2 (FIG. 7). Therefore, NeuroD1
and Dlx2 together can efficiently reprogram NG2 cells into neuronal
cells in the mouse brain in vivo. It is noted that the human NG2
promoter is not a specific promoter.
[0071] The following was performed to investigate whether NG2 cells
in the striatum were converted into GABAergic neurons, as found in
NG2 cell cultures. GABA and GAD67 immunostaining were performed,
which confirmed that many NG2-converted neurons were indeed
GABAergic neurons (FIG. 6F-G; 61.2.+-.3.6% infected cells were
GABA+; n=4 animals; 21 DPI). To test whether NG2-converted neurons
were functionally connected with other neurons, brain slice
recordings were performed at 1-1.5 months after NeuroD1+Dlx2 viral
injection. The NG2-converted neurons exhibited robust spontaneous
synaptic events (FIG. 6H; frequency, 6.8.+-.1.3 Hz; amplitude,
13.3.+-.0.9 pA; HP=-70 mV; n=16; 30-45 DPI). These results
demonstrate that striatal NG2 cells can be reprogrammed into
functional GABAergic neurons in situ after ectopic expression of
NeuroD1 and Dlx2.
Regional Influence on the Subtypes of GABAergic Neurons
[0072] The subtypes of in vivo NG2-converted GABAergic neurons were
further characterized using a series of GABAergic neuron markers
(FIG. 8). In the striatum, medium spiny neurons are projecting
neurons, not interneurons, but they are GABAergic neurons
(Gangarossa et al., Front Neural Circuits, 7:22 (2013)). A
significant proportion of NG2-converted neurons in the striatum
were DARPP32-positive medium spiny neurons (FIG. 8A, 40.6.+-.2.8%).
Because medium spiny neurons are mostly sensitive to the toxic
effects in Huntington's disease, this in vivo reprogramming method
can be used as a new therapy to treat Huntington's disease by
regenerating medium spiny neurons from internal glial cells. There
also was a significant number of NG2-converted neurons
immunopositive for PV (FIG. 8B), but rarely positive for CCK8 (FIG.
8C-D). Quantitatively, about 19.9.+-.1.9% of NG2-converted neurons
were PV.sup.+ GABAergic neurons and 9.3.+-.2.3% neurons were
SST.sup.+ (FIG. 8F, n=3 animals), consistent with previous report
that PV and SST neurons are the two major subtypes of interneurons
in the striatum (Marin et al., J. Neurosci., 20:6063-6076 (2000)).
Functional analysis with brain slice recordings revealed that some
NG2-converted neurons were capable of firing fast-spiking action
potentials (FIG. 8G), indicating that they are likely PV
interneurons. These results demonstrate that striatal NG2 cells can
be reprogrammed in situ into DARPP32.sup.+ and PV.sup.+ GABAergic
neurons by ectopic expression of Dlx2 and NeuroD1 together.
[0073] The following was performed to investigate whether NG2 cells
in different brain regions are reprogrammed into different subtypes
of GABAergic neurons when using the same transcription factors
NeuroD1+Dlx2. The same rAAV used in the striatum
(NG2::Cre+FLEx-NeuroD1+FLEx-Dlx2) was injected into mouse
prefrontal cortex, where the subtypes of GABAergic neurons are
different from the striatum. As a control experiment, expression of
mCherry alone (NG2::Cre+FLEx-mCherry) infected mainly NG2 cells in
the prefrontal cortex (FIG. 9A, 77.5.+-.1.3% infected cells were
NG2+; n=3 animals; see, also, FIG. 11). In contrast, ectopic
expression of Dlx2 and NeuroD1 in the NG2 cells of prefrontal
cortex reprogrammed most of the NG2 cells into neurons (FIG. 9B-C;
72.+-.1.1% mCherry.sup.+ cells were also NeuN.sup.+; n=3 animals;
21 DPI). Importantly, the majority of NG2-converted neurons also
were immunopositive for GABA or GAD67 (FIG. 9D-E; 69.3.+-.8%
mCherry.sup.+ cells were also GAD67.sup.+; n=3 animals; 21 DPI),
suggesting that they were GABAergic neurons. Moreover, brain slice
recordings showed robust spontaneous synaptic events in the
cortical NG2-converted neurons (FIG. 9F; frequency, 4.5.+-.1.7 Hz;
amplitude, 14.6.+-.2.1 pA; n=7; 30-45 DPI), indicating that the
NG2-converted neurons were functionally integrated into local
neural circuits. Therefore, similar to striatal NG2 cells, cortical
NG2 cells can be reprogrammed into functional GABAergic neurons by
ectopic expression of Dlx2 and NeuroD1 together. However, different
from the striatum where a large proportion of NG2-reprogrammed
cells were DARPP32-positive medium spiny neurons, cortical NG2
cells were found to be reprogrammed into mainly CCK8 (39.7.+-.2.2%,
n=3 animals) or PV-positive neurons (26.3.+-.3.4%, n=3 animals)
(FIGS. 10B, 10D, and 10F), while fewer neurons were SST- or
NPY-positive (FIGS. 10A and 10C). No DARPP32-positive cells were
detected among cortical NG2-converted cell population (FIG. 10E-F).
Patch-clamp recordings showed that cortical NG2-converted neurons
also were capable of firing fast-spiking action potentials or lower
frequency action potentials (FIG. 10G), confirming a mixture of
different subtypes of GABAergic neurons converted from cortical NG2
cells. These results demonstrate that striatal and cortical NG2
cells can be reprogrammed into different subtypes of GABAergic
neurons after expressing the same transcription factors Dlx2 and
NeuroD1, indicating that either intrinsic lineage trace inside the
regional glial cells or local environmental factors may regulate
the fate choice of glia-neuron conversion.
Example 2--Regenerating Medium Spiny Neurons to Treat Huntington's
Disease
[0074] A Huntington's disease (HD) mouse model, R6/2 transgenic
mice, which carries 120 CAG repeats from human HD gene and exhibit
disease onset at about 8-12 weeks of age is obtained. NeuroD1+Dlx2
AAV is injected into these HD mice at age of 6, 8, 10, and 12 weeks
old to confirm that the in vivo reprogramming technology described
herein extends the life of HD mice and improves the function of
these HD mice. Injecting NeuroD1 and Dlx2 into the striatum of HD
mice can have the ability to regenerate functional GABAergic
neurons including medium spiny neurons, which in turn can increase
the life span and rescue at least some of the functional deficits
of HD mice.
Example 3--In Situ Conversion of Glial Cells into GABAergic Neurons
Inside Brains
[0075] AAV5 viral vectors were produced to express NeuroD1 and
Dlx2. The AAV5 expressing NeuroD1 and Dlx2 viral vectors were
injected into R6/2 mice, a mouse model for Huntington's disease.
Following injection of AAV5 expressing NeuroD1 and Dlx2, astrocytes
of the striatum generated new neurons in the Huntington's disease
model R6/2 mice (FIGS. 14A-F). Most of the NeuroD1+Dlx2 converted
neurons were GABAergic neurons (FIGS. 15A-B).
[0076] In addition to AAV5 viral vectors, the capability of AAV9
viral vectors for in vivo cell conversion was confirmed. An AAV9
viral vector was used to express NeuroD1 and Dlx2 in astrocytes
under the control of astrocyte promoter GFAP. The AAV9 viral
vectors expressing NeuroD1 and Dlx2 were able to convert astrocytes
into neurons (FIG. 16A-D). Furthermore, immunostaining with a
GABAergic neuron marker (GAD67) demonstrated that the majority of
NeuroD1/Dlx2-converted neurons were GABAergic neurons (FIG. 17A-B).
Among the GABAergic neurons, some of the NeuroD1/Dlx2-converted
neurons were DARPP32-positive medium spiny neurons, which are often
vulnerable in Huntington's disease. These results demonstrate that
adeno-associated viral vectors such as AAV5 or AAV9 can be designed
to express NeuroD1 and Dlx2 and that such designed vectors can be
used to convert glial cells into GABAergic neurons, including
DARPP32-positive medium spiny neurons, thereby treating
Huntington's disease.
Other Embodiments
[0077] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
21356PRTHomo sapiens 1Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met
Gly Glu Pro Gln Pro1 5 10 15Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys
Leu Ser Ser Gln Asp Glu 20 25 30Glu His Glu Ala Asp Lys Lys Glu Asp
Asp Leu Glu Ala Met Asn Ala 35 40 45Glu Glu Asp Ser Leu Arg Asn Gly
Gly Glu Glu Glu Asp Glu Asp Glu 50 55 60Asp Leu Glu Glu Glu Glu Glu
Glu Glu Glu Glu Asp Asp Asp Gln Lys65 70 75 80Pro Lys Arg Arg Gly
Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu 85 90 95Glu Arg Phe Lys
Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110Arg Met
His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120
125Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg
130 135 140Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg
Ser Gly145 150 155 160Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr
Leu Cys Lys Gly Leu 165 170 175Ser Gln Pro Thr Thr Asn Leu Val Ala
Gly Cys Leu Gln Leu Asn Pro 180 185 190Arg Thr Phe Leu Pro Glu Gln
Asn Gln Asp Met Pro Pro His Leu Pro 195 200 205Thr Ala Ser Ala Ser
Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro 210 215 220Gly Leu Pro
Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe225 230 235
240His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe
245 250 255Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp
Gly Pro 260 265 270Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser
Phe Lys His Glu 275 280 285Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala
Phe Thr Met His Tyr Pro 290 295 300Ala Ala Thr Leu Ala Gly Ala Gln
Ser His Gly Ser Ile Phe Ser Gly305 310 315 320Thr Ala Ala Pro Arg
Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe 325 330 335Asp Ser His
Ser His His Glu Arg Val Met Ser Ala Gln Leu Asn Ala 340 345 350Ile
Phe His Asp 3552328PRTHomo sapiens 2Met Thr Gly Val Phe Asp Ser Leu
Val Ala Asp Met His Ser Thr Gln1 5 10 15Ile Ala Ala Ser Ser Thr Tyr
His Gln His Gln Gln Pro Pro Ser Gly 20 25 30Gly Gly Ala Gly Pro Gly
Gly Asn Ser Ser Ser Ser Ser Ser Leu His 35 40 45Lys Pro Gln Glu Ser
Pro Thr Leu Pro Val Ser Thr Ala Thr Asp Ser 50 55 60Ser Tyr Tyr Thr
Asn Gln Gln His Pro Ala Gly Gly Gly Gly Gly Gly65 70 75 80Gly Ser
Pro Tyr Ala His Met Gly Ser Tyr Gln Tyr Gln Ala Ser Gly 85 90 95Leu
Asn Asn Val Pro Tyr Ser Ala Lys Ser Ser Tyr Asp Leu Gly Tyr 100 105
110Thr Ala Ala Tyr Thr Ser Tyr Ala Pro Tyr Gly Thr Ser Ser Ser Pro
115 120 125Ala Asn Asn Glu Pro Glu Lys Glu Asp Leu Glu Pro Glu Ile
Arg Ile 130 135 140Val Asn Gly Lys Pro Lys Lys Val Arg Lys Pro Arg
Thr Ile Tyr Ser145 150 155 160Ser Phe Gln Leu Ala Ala Leu Gln Arg
Arg Phe Gln Lys Thr Gln Tyr 165 170 175Leu Ala Leu Pro Glu Arg Ala
Glu Leu Ala Ala Ser Leu Gly Leu Thr 180 185 190Gln Thr Gln Val Lys
Ile Trp Phe Gln Asn Arg Arg Ser Lys Phe Lys 195 200 205Lys Met Trp
Lys Ser Gly Glu Ile Pro Ser Glu Gln His Pro Gly Ala 210 215 220Ser
Ala Ser Pro Pro Cys Ala Ser Pro Pro Val Ser Ala Pro Ala Ser225 230
235 240Trp Asp Phe Gly Val Pro Gln Arg Met Ala Gly Gly Gly Gly Pro
Gly 245 250 255Ser Gly Gly Ser Gly Ala Gly Ser Ser Gly Ser Ser Pro
Ser Ser Ala 260 265 270Ala Ser Ala Phe Leu Gly Asn Tyr Pro Trp Tyr
His Gln Thr Ser Gly 275 280 285Ser Ala Ser His Leu Gln Ala Thr Ala
Pro Leu Leu His Pro Thr Gln 290 295 300Thr Pro Gln Pro His His His
His His His His Gly Gly Gly Gly Ala305 310 315 320Pro Val Ser Ala
Gly Thr Ile Phe 325
* * * * *