U.S. patent application number 14/725888 was filed with the patent office on 2016-03-03 for compositions and methods to treat latent viral infections.
This patent application is currently assigned to THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY. The applicant listed for this patent is THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY. Invention is credited to STEPHEN R. QUAKE, JIANBIN WANG.
Application Number | 20160060655 14/725888 |
Document ID | / |
Family ID | 53366336 |
Filed Date | 2016-03-03 |
United States Patent
Application |
20160060655 |
Kind Code |
A1 |
QUAKE; STEPHEN R. ; et
al. |
March 3, 2016 |
COMPOSITIONS AND METHODS TO TREAT LATENT VIRAL INFECTIONS
Abstract
Viral infection is a persistent cause of human disease. Guided
nuclease systems target the genomes of viral infections, rendering
the viruses incapacitated.
Inventors: |
QUAKE; STEPHEN R.; (Palo
Alto, CA) ; WANG; JIANBIN; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR
UNIVERSITY |
Palo Alto |
CA |
US |
|
|
Assignee: |
THE BOARD OF TRUSTEES OF THE LELAND
STANFORD JUNIOR UNIVERSITY
Palo Alto
CA
|
Family ID: |
53366336 |
Appl. No.: |
14/725888 |
Filed: |
May 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62029072 |
Jul 25, 2014 |
|
|
|
62005395 |
May 30, 2014 |
|
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Current U.S.
Class: |
424/94.61 ;
435/199; 435/461; 435/463; 536/23.2 |
Current CPC
Class: |
Y02A 50/385 20180101;
Y02A 50/393 20180101; C12N 9/16 20130101; C12Y 301/00 20130101;
C12N 15/86 20130101; A61P 31/12 20180101; A61P 31/20 20180101; C12N
15/1133 20130101; A61P 43/00 20180101; C12N 2330/51 20130101; A61P
31/14 20180101; C12N 2810/60 20130101; A61P 31/18 20180101; A61K
48/005 20130101; C12N 2820/60 20130101; C12N 9/22 20130101; Y02A
50/387 20180101; Y02A 50/30 20180101; Y02A 50/467 20180101; Y02A
50/463 20180101; C12N 15/907 20130101; C12N 2310/20 20170501; A61P
31/22 20180101; A61K 38/00 20130101; Y02A 50/465 20180101 |
International
Class: |
C12N 15/90 20060101
C12N015/90; C12N 9/22 20060101 C12N009/22 |
Claims
1. A method for treating a viral infection, the method comprising:
introducing into a cell a nuclease and a sequence-specific
targeting moiety; targeting the nuclease to a viral nucleic acid by
means of the sequence-specific targeting moiety; and cleaving the
viral nucleic acid with the nuclease without interfering with a
host genome.
2. The method of claim 1, wherein the nuclease is one selected from
the group consisting of a zinc-finger nuclease, a transcription
activator-like effector nuclease, and a meganuclease.
3. The method of claim 1, wherein the nuclease comprises Cas9
endonuclease and the sequence-specific targeting moiety comprises a
guide RNA.
4. The method of claim 1, wherein the viral infection is latent and
the viral nucleic acid is integrated into the host genome.
5. The method of claim 1, wherein cleaving includes making a double
strand break in the viral nucleic acid.
6. The method of claim 1, wherein the host is a living human
subject and the steps are performed in vivo.
7. The method of claim 1, further comprising inserting a
polynucleotide into the viral nucleic acid.
8. The method of claim 1, wherein the viral nucleic acid is from a
virus that is selected from the group consisting of Adenovirus,
Herpes simplex, type 1, Herpes simplex, type 2, Varicella-zoster
virus, Epstein-barr virus, Human cytomegalovirus, Human
herpesvirus, type 8, Human papillomavirus, BK virus, JC virus,
Smallpox, Hepatitis B virus, Human bocavirus, Parvovirus B19, Human
astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus,
poliovirus, rhinovirus, Severe acute respiratory syndrome virus,
Hepatitis C virus, yellow fever virus, dengue virus, West Nile
virus, Rubella virus, Hepatitis E virus, Human immunodeficiency
virus (HIV), Influenza virus, Guanarito virus, Junin virus, Lassa
virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever
virus, Ebola virus, Marburg virus, Measles virus, Mumps virus,
Parainfluenza virus, Respiratory syncytial virus, Human
metapneumovirus, Hendra virus, Nipah virus, Rabies virus, Hepatitis
D, Rotavirus, Orbivirus, Coltivirus, and Banna virus.
9. The method of claim 1, wherein introducing the nuclease and
sequence-specific targeting moiety comprises introducing into the
cell a viral vector that encodes the nuclease and sequence-specific
targeting moiety.
10. The method of claim 9, wherein the viral vector is selected
from the group consisting of retrovirus, lentivirus, adenovirus,
herpesvirus, poxvirus, alphavirus, vaccinia virus and
adeno-associated viruses.
11. The method of claim 1, wherein introducing the nuclease and
sequence-specific targeting moiety comprises introducing into the
cell a non-viral vector.
12. The method of claim 11, wherein the non-viral vector is
selected from the group consisting of a nanoparticle, a cationic
lipid, a cationic polymer, metallic nanoparticle, a nanorod, a
liposome, microbubbles, a cell-penetrating peptide, and a
liposphere.
13. The method of claim 11, wherein the non-viral vector comprises
polyethyleneglycol (PEG).
14. The method of claim 11, further comprising applying energy to
the vector.
15. The method of claim 11, wherein the energy is introduced via
ultrasound or electroporation.
16. A composition for treatment of a viral infection, the
composition comprising: a nuclease; and a sequence-specific
targeting moiety that targets the nuclease to a viral nucleic acid
in vivo within a host cell thereby causing the nuclease to cleave
the viral nucleic acid without interfering with host nucleic
acid.
17. The composition of claim 16, wherein the nuclease is a Cas9
endonuclease and the sequence-specific binding module comprises a
guide RNA that specifically targets a portion of a viral
genome.
18. The composition of claim 17, wherein the Cas9 endonuclease and
the guide RNA are co-expressed in a host cell infected by a
virus.
19. The composition of claim 16, wherein the nuclease is one
selected from the list consisting of a zinc-finger nuclease, a
transcription activator-like effector nuclease, and a
meganuclease.
20. The composition of claim 16, wherein the viral nucleic acid is
from a virus that is selected from the group consisting of herpes
simplex virus (HSV)-1, HSV-2, varicella zoster virus (VZV),
cytomegalovirus (CMV), human herpesvirus (HHV)-6, HHV-7, Kaposi's
sarcoma-associated herpesvirus (KSHV), JC virus, BK virus,
parvovirus b19, adeno-associated virus (AAV), and adenovirus.
21. A composition for treatment of a viral infection, the
composition comprising: nucleic acid encoding: a nuclease, and a
sequence-specific targeting moiety that targets the nuclease to
viral nucleic acid thereby causing the nuclease to cleave the viral
nucleic acid without interfering with host nucleic acid.
22. The composition of claim 21, wherein the sequence-specific
targeting moiety comprises a guide RNA.
23. The composition of claim 22, wherein the guide RNA is
complementary to a portion of a viral genome.
24. The composition of claim 23, wherein the guide RNA is designed
to cause the nuclease to cleave the viral genome within a feature
that is necessary for viral function.
25. The composition of claim 24, wherein the feature is one
selected from the list consisting of: a viral replication origin, a
terminal repeat, a replication factor binding site, a promoter, a
coding sequence, and a repetitive region.
26. The composition of claim 21, wherein the nucleic acid is
provided within a delivery vector.
27. The composition of claim 26, wherein the delivery vector
comprises an adeno-associated virus.
28. The composition of claim 27, wherein the delivery vector
comprises on selected from the group consisting of retrovirus,
lentivirus, adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia
virus, a nanoparticle, a cationic lipid, a cationic polymer, a
metallic nanoparticle, a nanorod, a liposome, microbubbles,
cell-penetrating peptide, a liposphere, and polyethyleneglycol
(PEG).
29. The composition of claim 21, wherein the nuclease is a Cas9
endonuclease.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims priority to, and the benefit of,
both U.S. Provisional Patent Application Ser. No. 62/005,395, filed
May 30, 2014, and U.S. Provisional Patent Application Ser. No.
62/029,072, filed Jul. 25, 2014, the contents of which are
incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention generally relates to compositions and methods
for selectively treating viral infections using a guided nuclease
system.
BACKGROUND
[0003] Viral infections are a significant medical problem. Various
antiviral treatments are available but they generally are directed
to interrupting the replicating cycle of the virus. Thus, a
particularly difficult problem is latent viral infection, as there
is no effective treatment to eradicate the virus from host cells.
Since latent infection can evade immune surveillance and reactivate
the lytic cycle at any time, there is a persistent risk throughout
the life. The majority of antiviral drug development has been
focused on protein targets and such approaches have not been
successful in eradicating the virus.
[0004] One example of a latent viral infection that is a particular
problem is the herpesviridae virus family. Herpes is one of the
most widespread human pathogens, with more than 90% of adults
having been infected with at least one of the eight subtypes of
herpes viruse. Latent infection persists in most people; and about
16% of Americans between the ages of 14 and 49 are infected with
genital herpes, making it one of the most common sexually
transmitted diseases. Due to latency, there is no cure for genital
herpes or for herpes simplex virus type 2 (HSV-2). Once infected, a
host carries the herpes virus indefinitely, even when not
expressing symptoms. Similarly, human papillomavirus, or HPV is a
common virus in the human population, where more than 75% of women
and men will have this type of infection at one point in their
life. High-risk oncogenic HPV types are able to integrate into the
DNA of the cell that can result in cancer, specifically cervical
cancer. Similar to the herpesviridae virus family, HPV may remain
latent.
[0005] The Epstein-Barr virus (EBV), also called human herpesvirus
4 (HHV-4) is another common virus in humans. Epstein-Barr is known
as the cause of infectious mononucleosis (glandular fever), and is
also associated with particular forms of cancer, such as Hodgkin's
lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma, and
conditions associated with human immunodeficiency virus (HIV) such
as hairy leukoplakia and central nervous system lymphomas. There is
evidence that infection with the virus is associated with a higher
risk of certain autoimmune diseases, especially dermatomyositis,
systemic lupus erythematosus, rheumatoid arthritis, Sjogren's
syndrome, and multiple sclerosis. During latency, the EBV genome
circularizes and resides in the cell nucleus as episomes. To date,
however, no EBV vaccine or treatment exists.
[0006] Viruses, such as the herpesviridae virus family, including
EBV, and HPV have the ability to lie dormant within a cell
indefinitely and not be fully eradicated even after treatment. The
result is that the virus can reactivate and begin producing large
amounts of viral progeny without the host being infected by any new
outside virus. In the latent state, the viral genome persists
within the host cells as episomes; stabilized and floating in the
cytoplasm or nucleus. For these latent viruses, it has not been
possible to find therapeutic approaches which completely eradicate
such infections.
SUMMARY
[0007] The invention provides methods for selectively treating
viral infections using a guided nuclease system. Methods of the
invention may be used to remove viral or other foreign genetic
material from a host organism, without interfering the integrity of
the host's genetic material. A nuclease may be used to target viral
nucleic acid, thereby interfering with viral replication or
transcription or even excising the viral genetic material from the
host genome. The nuclease may be specifically targeted to remove
only the viral nucleic acid without acting on host material either
when the viral nucleic acid exists as a particle within the cell or
when it is integrated into the host genome. Targeting the viral
nucleic acid can be done using a sequence-specific moiety such as a
guide RNA that targets viral genomic material for destruction by
the nuclease and does not target the host cell genome. In some
embodiments, a CRISPR/Cas9 nuclease and guide RNA (gRNA) that
together target and selectively edit or destroy viral genomic
material is used. The CRISPR (clustered regularly interspaced short
palindromic repeats) is a naturally-occurring element of the
bacterial immune system that protects bacteria from phage
infection. The guide RNA localizes the CRISPR/Cas9 complex to a
viral target sequence. Binding of the complex localizes the Cas9
endonuclease to the viral genomic target sequence causing breaks in
the viral genome. Other nuclease systems can be used including, for
example, zinc finger nucleases, transcription activator-like
effector nucleases (TALENs), meganucleases, or any other system
that can be used to degrade or interfere with viral nucleic acid
without interfering with the regular function of the host's genetic
material.
[0008] In certain aspects, the invention provides a method for
treating a viral infection. The method includes introducing into a
cell a nuclease and a sequence-specific targeting moiety. The
nuclease is targeted to viral nucleic acid by means of the
sequence-specific targeting moiety and the nuclease cleaves the
viral nucleic acid without interfering with a host genome. The
nuclease may be, for example, a zinc-finger nuclease, a
transcription activator-like effector nuclease, and a meganuclease.
In a preferred embodiment, the nuclease is a Cas9 endonuclease and
the sequence-specific targeting moiety comprises a guide RNA. The
cleaving step can make one or more single or double stranded breaks
in the viral nucleic acid. The method may further include inserting
a polynucleotide or re-joining the cleaved ends with a piece of the
viral nucleic acid removed. The host may be a living subject such
as a human patient and the steps may be performed in vivo.
[0009] The method may be used to target viral nucleic acid in any
form or at any stage in the viral life cycle. For example, the
method may be used to digest viral RNA or DNA. The targeted viral
nucleic acid may be present in the host cell as independent
particles. In a preferred embodiment, the viral infection is latent
and the viral nucleic acid is integrated into the host genome. Any
suitable viral nucleic acid may be targeted for cleavage and
digestion. In certain embodiments, the targeted virus includes one
or more of herpes simplex virus (HSV)-1, HSV-2, varicella zoster
virus (VZV), cytomegalovirus (CMV), human herpesvirus (HHV)-6,
HHV-7, Kaposi's sarcoma-associated herpesvirus (KSHV), JC virus, BK
virus, parvovirus b19, adeno-associated virus (AAV), and
adenovirus. In some embodiments, the targeted virus or viruses
include one or more of Adenovirus, Herpes simplex, type 1, Herpes
simplex, type 2, Varicella-zoster virus, Epstein-barr virus, Human
cytomegalovirus, Human herpesvirus, type 8, Human papillomavirus,
BK virus, JC virus, Smallpox, Hepatitis B virus, Human bocavirus,
Parvovirus B19, Human astrovirus, Norwalk virus, coxsackievirus,
hepatitis A virus, poliovirus, rhinovirus, Severe acute respiratory
syndrome virus, Hepatitis C virus, yellow fever virus, dengue
virus, West Nile virus, Rubella virus, Hepatitis E virus, Human
immunodeficiency virus (HIV), Influenza virus, Guanarito virus,
Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo
hemorrhagic fever virus, Ebola virus, Marburg virus, Measles virus,
Mumps virus, Parainfluenza virus, Respiratory syncytial virus,
Human metapneumovirus, Hendra virus, Nipah virus, Rabies virus,
Hepatitis D, Rotavirus, Orbivirus, Coltivirus, and Banna virus.
[0010] The nuclease and sequence-specific targeting moiety may be
introduced into the cell using a vector. For example, a viral
vector that encodes the nuclease and sequence-specific targeting
moiety may be used. The viral vector may be retrovirus, lentivirus,
adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia virus or
adeno-associated viruses. In some embodiments, a non-viral vector
is used. A suitable non-viral vector may include, for example, a
nanoparticle, a cationic lipid, a cationic polymer, metallic
nanoparticle, a nanorod, a liposome, microbubbles, a
cell-penetrating peptide, a liposphere, polyethyleneglycol (PEG).
The cell may be prompted to take up the vector by, e.g., ultrasound
or electroporation.
[0011] Aspects of the invention provide a composition for treatment
of a viral infection. The composition includes a nuclease and a
sequence-specific targeting moiety that targets the nuclease to
viral nucleic acid in vivo within a host cell thereby causing the
nuclease to cleave the viral nucleic acid without interfering with
host nucleic acid. In certain embodiments, the nuclease is a Cas9
endonuclease and the sequence-specific binding module comprises a
guide RNA that specifically targets a portion of a viral genome.
The Cas9 endonuclease and the guide RNA may be co-expressed in a
host cell infected by a virus. In some embodiments, the nuclease is
one selected from the list consisting of a zinc-finger nuclease, a
transcription activator-like effector nuclease, and a
meganuclease.
[0012] The viral nucleic acid to be cleaved may include one or more
of, e.g., herpes simplex virus (HSV)-1, HSV-2, varicella zoster
virus (VZV), cytomegalovirus (CMV), human herpesvirus (HHV)-6,
HHV-7, Kaposi's sarcoma-associated herpesvirus (KSHV), JC virus, BK
virus, parvovirus b19, adeno-associated virus (AAV), and
adenovirus, or others.
[0013] In some aspects, the invention provides a composition for
treatment of a viral infection. The composition includes nucleic
acid that encodes a nuclease and a sequence-specific targeting
moiety that targets the nuclease to viral nucleic acid thereby
causing the nuclease to cleave the viral nucleic acid without
interfering with host nucleic acid. In some embodiments, the
sequence-specific targeting moiety uses a guide RNA, which may be
complementary to a portion of a viral genome. The guide RNA may be
designed to cause the nuclease to cleave the viral genome within a
feature that is necessary for viral function. The feature may be,
for example, a viral replication origin, a terminal repeat, a
replication factor binding site, a promoter, a coding sequence, or
a repetitive region. The nucleic acid is provided within a delivery
vector which may be a viral vector such as an adeno-associated
virus. The vector could include any of retrovirus, lentivirus,
adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia virus, a
nanoparticle, a cationic lipid, a cationic polymer, a metallic
nanoparticle, a nanorod, a liposome, microbubbles, cell-penetrating
peptide, a liposphere, or polyethyleneglycol (PEG).
[0014] Methods and compositions of the invention may be used to
deliver a CRISPR/gRNA/Cas9 complex to a cell (including entire
tissues) that is infected by a virus. a guide RNA may be designed
to target multiple sites on the viral genome in order to disrupt
viral nucleic acid and reduce the chance that it will functionally
recombine. The CRISPR/gRNA/Cas9 complexes of the invention can be
delivered by viral, non-viral or other methods to effectuate
transfection. CRISPR/gRNA/Cas9 complexes are preferably designed to
target viral genomic material and not genomic material of the host.
In some embodiments, the targeted viral nucleic acid is associated
with a virus that causes latent infection. Latent viruses may be,
for example, human immunodeficiency virus, human T-cell leukemia
virus, Epstein-Barr virus, human cytomegalovirus, human
herpesviruses 6 and 7, herpes simplex virus types 1 and 2,
varicella-zoster virus, measles virus, or human papovaviruses.
Aspects of the invention allow for CRISPR/gRNA/Cas9 complexes to be
designed to target any virus, latent or active.
[0015] The presented methods allow for viral genome editing or
destruction, which results in the inability of the virus to
proliferate and/or induces apoptosis in infected cells, with no
observed cytotoxicity to non-infected cells. A CRISPR/gRNA/Cas9
complex is designed to selectively target viral genomic material
(DNA or RNA), delivering the CRISPR/gRNA/Cas9 complex to a cell
containing the viral genome, and cutting the viral genome in order
to incapacitate the virus. A viral infection can thus be treated by
targeted disruption of viral genomic function or by digestion of
viral nucleic acid via one or multiple breaks caused by targeting
sites for endonuclease action in the viral genome. In some
embodiments, methods of the invention may be used for transfection
of a host cell with CRISPR/gRNA/Cas9 to completely suppressed cell
proliferation and/or induce apoptosis in infected cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-1C represent EBV-targeting CRISPR/Cas9 designs.
(FIG. 1A) Scheme of CRISPR/Cas plasmids, adapted from Cong L et al.
(2013) Multiplex Genome Engineering Using CRISPR/Cas Systems.
Science 339:819-823. (FIG. 1B) Effect of oriP on transfection
efficiency in Raji cells. Both Cas9 and Cas9-oriP plasmids have a
scrambled guide RNA. (FIG. 1C) CRISPR guide RNA targets along the
EBV reference genome. Green, red and blue represent three different
target sequence categories.
[0017] FIGS. 2A-2F represent CRISPR/Cas9 induced large deletions.
(FIG. 2A) Genome context around guide RNA sgEBV2 and PCR primer
locations. (FIG. 2B) Large deletion induced by sgEBV2. Lane 1-3 are
before, 5 days after, and 7 days after sgEBV2 treatment,
respectively. (FIG. 2C) Genome context around guide RNA sgEBV3/4/5
and PCR primer locations. (FIG. 2D) Large deletions induced by
sgEBV3/5 and sgEBV4/5. Lane 1 and 2 are 3F/5R PCR amplicons before
and 8 days after sgEBV3/5 treatment. Lane 3 and 4 are 4F/5R PCR
amplicons before and 8 days after sgEBV4/5 treatment. (FIGS. 2E and
F) Sanger sequencing confirmed genome cleavage and repair ligation
8 days after sgEBV3/5 (FIG. 2E) and sgEBV4/5 (FIG. 2F) treatment.
Blue and white background highlights the two ends before repair
ligation.
[0018] FIGS. 3A-3M represent cell proliferation arrest with EBV
genome destruction. (FIG. 3A) Cell proliferation curves after
different CRISPR treatments. Five independent sgEBV1-7 treatments
are shown here. (FIGS. 3B-D) Flow cytometry scattering signals
before (FIG. 3B), 5 days after (FIG. 3C) and 8 days after (FIG. 3D)
sgEBV1-7 treatments. (FIG. 3E-G) Annexin V Alexa647 and DAPI
staining results before (FIG. 3E), 5 days after (FIG. 3F) and 8
days after (FIG. 3G) sgEBV1-7 treatments. Blue and red correspond
to subpopulation P3 and P4 in (FIGS. 3B-D). (FIGS. 3H and I)
Microscopy revealed apoptotic cell morphology after sgEBV1-7
treatment. (FIGS. 3J-M) Nuclear morphology before (FIG. 3J) and
after (FIGS. 3K-M) sgEBV1-7 treatment.
[0019] FIGS. 4A-4E represent EBV load quantitation after CRISPR
treatment. (FIG. 4A) EBV load after different CRISPR treatments by
digital PCR. Cas9 and Cas9-oriP had two replicates, and sgEBV1-7
had 5 replicates. (FIGS. 4B and C) Microscopy of captured single
cells for whole-genome amplification. (FIG. 4D) Histogram of EBV
quantitative PCR Ct values from single cells before treatment.
(FIG. 4E) Histogram of EBV quantitative PCR Ct values from single
live cells 7 days after sgEBV1-7 treatment. Red dash lines in (FIG.
4D) and (FIG. 4E) represent Ct values of one EBV genome per
cell.
[0020] FIG. 5 represents SURVEYOR assay of EBV CRISPR. Lane 1: NEB
100 bp ladder; Lane 2: sgEBV1 control; Lane 3: sgEBV1; Lane 4:
sgEBV5 control; Lane 5: sgEBV5; Lane 6: sgEBV7 control; Lane 7:
sgEBV7; Lane 8: sgEBV4.
[0021] FIG. 6 shows CRISPR cytotoxicity test with EBV-negative
Burkitt's lymphoma DG-75.
[0022] FIG. 7 represents CRISPR cytotoxicity test with primary
human lung fibroblast IMR-90.
[0023] FIG. 8 shows the use of ZFNs.
[0024] FIG. 9 diagrams a method of the invention.
[0025] FIG. 10 is a map of an HBV genome.
[0026] FIG. 11 shows the results of delivering a viral
treatment.
[0027] FIG. 12 shows a composition for treating a viral
infection.
DETAILED DESCRIPTION
[0028] The invention generally relates to compositions and methods
for selectively treating viral infections using a guided nuclease
system. Methods of the invention are used to incapacitate or
disrupt viral nucleic acid within a cell through nuclease activity
such as single- or double-stranded breaks, cleavage, digestion, or
editing. Methods of the invention may be used for systematically
causing large or repeated deletions in the genome, reducing the
probability of reconstructing the full genome.
i. Treating Infected Cell
[0029] FIG. 9 diagrams a method of treating a cell infected with a
virus. Methods of the invention are applicable to in vivo treatment
of patients and may be used to remove any viral genetic material
such as genes of virus associated with a latent viral infection.
Methods may be used in vitro, e.g., to prepare or treat a cell
culture or cell sample. When used in vivo, the cell may be any
suitable germ line or somatic cell and compositions of the
invention may be delivered to specific parts of a patient's body or
be delivered systemically. If delivered systemically, it may be
preferable to include within compositions of the invention
tissue-specific promoters. For example, if a patient has a latent
viral infection that is localized to the liver, hepatic
tissue-specific promotors may be included in a plasmid or viral
vector that codes for a targeted nuclease.
[0030] FIG. 12 shows a composition for treating a viral infection
according to certain embodiments. The composition preferably
includes a vector (which may be a plasmid, linear DNA, or a viral
vector) that codes for a nuclease and a targeting moiety (e.g., a
gRNA) that targets the nuclease to viral nucleic acid. The
composition may optionally include one or more of a promoter,
replication origin, other elements, or combinations thereof as
described further herein.
ii. Nuclease
[0031] Methods of the invention include using a programmable or
targetable nuclease to specifically target viral nucleic acid for
destruction. Any suitable targeting nuclease can be used including,
for example, zinc-finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs), clustered regularly
interspaced short palindromic repeat (CRISPR) nucleases,
meganucleases, other endo- or exo-nucleases, or combinations
thereof. See Schiffer, 2012, Targeted DNA mutagenesis for the cure
of chronic viral infections, J Virol 88(17):8920-8936, incorporated
by reference.
[0032] CRISPR methodologies employ a nuclease, CRISPR-associated
(Cas9), that complexes with small RNAs as guides (gRNAs) to cleave
DNA in a sequence-specific manner upstream of the protospacer
adjacent motif (PAM) in any genomic location. CRISPR may use
separate guide RNAs known as the crRNA and tracrRNA. These two
separate RNAs have been combined into a single RNA to enable
site-specific mammalian genome cutting through the design of a
short guide RNA. Cas9 and guide RNA (gRNA) may be synthesized by
known methods. Cas9/guide-RNA (gRNA) uses a non-specific DNA
cleavage protein Cas9, and an RNA oligo to hybridize to target and
recruit the Cas9/gRNA complex. See Chang et al., 2013, Genome
editing with RNA-guided Cas9 nuclease in zebrafish embryos, Cell
Res 23:465-472; Hwang et al., 2013, Efficient genome editing in
zebrafish using a CRISPR-Cas system, Nat. Biotechnol 31:227-229;
Xiao et al., 2013, Chromosomal deletions and inversions mediated by
TALENS and CRISPR/Cas in zebrafish, Nucl Acids Res 1-11.
[0033] CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) is found in bacteria and is believed to protect the
bacteria from phage infection. It has recently been used as a means
to alter gene expression in eukaryotic DNA, but has not been
proposed as an anti-viral therapy or more broadly as a way to
disrupt genomic material. Rather, it has been used to introduce
insertions or deletions as a way of increasing or decreasing
transcription in the DNA of a targeted cell or population of cells.
See for example, Horvath et al., Science (2010) 327:167-170; Terns
et al., Current Opinion in Microbiology (2011) 14:321-327; Bhaya et
al Annu Rev Genet (2011) 45:273-297; Wiedenheft et al. Nature
(2012) 482:331-338); Jinek M et al. Science (2012) 337:816-821;
Cong L et al. Science (2013) 339:819-823; Jinek M et al. (2013)
eLife 2:e00471; Mali P et al. (2013) Science 339:823-826; Qi L S et
al. (2013) Cell 152:1173-1183; Gilbert L A et al. (2013) Cell
154:442-451; Yang H et al. (2013) Cell 154:1370-1379; and Wang H et
al. (2013) Cell 153:910-918).
[0034] In an aspect of the invention, the Cas9 endonuclease causes
a double strand break in at least two locations in the genome.
These two double strand breaks cause a fragment of the genome to be
deleted. Even if viral repair pathways anneal the two ends, there
will still be a deletion in the genome. One or more deletions using
the mechanism will incapacitate the viral genome. The result is
that the host cell will be free of viral infection.
[0035] In embodiments of the invention, nucleases cleave the genome
of the target virus. A nuclease is an enzyme capable of cleaving
the phosphodiester bonds between the nucleotide subunits of nucleic
acids. Endonucleases are enzymes that cleave the phosphodiester
bond within a polynucleotide chain. Some, such as Deoxyribonuclease
I, cut DNA relatively nonspecifically (without regard to sequence),
while many, typically called restriction endonucleases or
restriction enzymes, cleave only at very specific nucleotide
sequences. In a preferred embodiment of the invention, the Cas9
nuclease is incorporated into the compositions and methods of the
invention, however, it should be appreciated that any nuclease may
be utilized.
[0036] In preferred embodiments of the invention, the Cas9 nuclease
is used to cleave the genome. The Cas9 nuclease is capable of
creating a double strand break in the genome. The Cas9 nuclease has
two functional domains: RuvC and HNH, each cutting a different
strand. When both of these domains are active, the Cas9 causes
double strand breaks in the genome.
[0037] In some embodiments of the invention, insertions into the
genome can be designed to cause incapacitation, or altered genomic
expression. Additionally, insertions/deletions are also used to
introduce a premature stop codon either by creating one at the
double strand break or by shifting the reading frame to create one
downstream of the double strand break. Any of these outcomes of the
NHEJ repair pathway can be leveraged to disrupt the target gene.
The changes introduced by the use of the CRISPR/gRNA/Cas9 system
are permanent to the genome.
[0038] In some embodiments of the invention, at least one insertion
is caused by the CRISPR/gRNA/Cas9 complex. In a preferred
embodiment, numerous insertions are caused in the genome, thereby
incapacitating the virus. In an aspect of the invention, the number
of insertions lowers the probability that the genome may be
repaired.
[0039] In some embodiments of the invention, at least one deletion
is caused by the CRISPR/gRNA/Cas9 complex. In a preferred
embodiment, numerous deletions are caused in the genome, thereby
incapacitating the virus. In an aspect of the invention, the number
of deletions lowers the probability that the genome may be
repaired. In a highly-preferred embodiment, the CRISPR/Cas9/gRNA
system of the invention causes significant genomic disruption,
resulting in effective destruction of the viral genome, while
leaving the host genome intact.
[0040] TALENs uses a nonspecific DNA-cleaving nuclease fused to a
DNA-binding domain that can be to target essentially any sequence.
For TALEN technology, target sites are identified and expression
vectors are made. Linearized expression vectors (e.g., by Not1) may
be used as template for mRNA synthesis. A commercially available
kit may be use such as the mMESSAGE mMACHINE SP6 transcription kit
from Life Technologies (Carlsbad, Calif.). See Joung & Sander,
2013, TALENs: a widely applicable technology for targeted genome
editing, Nat Rev Mol Cell Bio 14:49-55.
[0041] TALENs and CRISPR methods provide one-to-one relationship to
the target sites, i.e. one unit of the tandem repeat in the TALE
domain recognizes one nucleotide in the target site, and the crRNA,
gRNA, or sgRNA of CRISPR/Cas system hybridizes to the complementary
sequence in the DNA target. Methods can include using a pair of
TALENs or a Cas9 protein with one gRNA to generate double-strand
breaks in the target. The breaks are then repaired via
non-homologous end joining or homologous recombination (HR).
[0042] FIG. 8 shows ZFN being used to cut viral nucleic acid.
Briefly, the ZFN method includes introducing into the infected host
cell at least one vector (e.g., RNA molecule) encoding a targeted
ZFN 305 and, optionally, at least one accessory polynucleotide.
See, e.g., U.S. Pub. 2011/0023144 to Weinstein, incorporated by
reference The cell includes target sequence 311. The cell is
incubated to allow expression of the ZFN 305, wherein a
double-stranded break 317 is introduced into the targeted
chromosomal sequence 311 by the ZFN 305. In some embodiments, a
donor polynucleotide or exchange polynucleotide 321 is introduced.
Swapping a portion of the viral nucleic acid with irrelevant
sequence can fully interfere transcription or replication of the
viral nucleic acid. Target DNA 311 along with exchange
polynucleotide 321 may be repaired by an error-prone non-homologous
end joining DNA repair process or a homology-directed DNA repair
process.
[0043] Typically, a ZFN comprises a DNA binding domain (i.e., zinc
finger) and a cleavage domain (i.e., nuclease) and this gene may be
introduced as mRNA (e.g., 5' capped, polyadenylated, or both). Zinc
finger binding domains may be engineered to recognize and bind to
any nucleic acid sequence of choice. See, e.g., Qu et al., 2013,
Zinc-finger-nucleases mediate specific and efficient excision of
HIV-1 proviral DAN from infected and latently infected human T
cells, Nucl Ac Res 41(16):7771-7782, incorporated by reference. An
engineered zinc finger binding domain may have a novel binding
specificity compared to a naturally-occurring zinc finger protein.
Engineering methods include, but are not limited to, rational
design and various types of selection. A zinc finger binding domain
may be designed to recognize a target DNA sequence via zinc finger
recognition regions (i.e., zinc fingers). See for example, U.S.
Pat. Nos. 6,607,882; 6,534,261 and 6,453,242, incorporated by
reference. Exemplary methods of selecting a zinc finger recognition
region may include phage display and two-hybrid systems, and are
disclosed in U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S.
Pat. No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No.
6,410,248; U.S. Pat. No. 6,140,466; U.S. Pat. No. 6,200,759; and
U.S. Pat. No. 6,242,568, each of which is incorporated by
reference.
[0044] A ZFN also includes a cleavage domain. The cleavage domain
portion of the ZFNs may be obtained from any suitable endonuclease
or exonuclease such as restriction endonucleases and homing
endonucleases. See, for example, Belfort & Roberts, 1997,
Homing endonucleases: keeping the house in order, Nucleic Acids Res
25(17):3379-3388. A cleavage domain may be derived from an enzyme
that requires dimerization for cleavage activity. Two ZFNs may be
required for cleavage, as each nuclease comprises a monomer of the
active enzyme dimer. Alternatively, a single ZFN may comprise both
monomers to create an active enzyme dimer. Restriction
endonucleases present may be capable of sequence-specific binding
and cleavage of DNA at or near the site of binding. Certain
restriction enzymes (e.g., Type IIS) cleave DNA at sites removed
from the recognition site and have separable binding and cleavage
domains. For example, the Type IIS enzyme FokI, active as a dimer,
catalyzes double-stranded cleavage of DNA, at 9 nucleotides from
its recognition site on one strand and 13 nucleotides from its
recognition site on the other. The FokI enzyme used in a ZFN may be
considered a cleavage monomer. Thus, for targeted double-stranded
cleavage using a FokI cleavage domain, two ZFNs, each comprising a
FokI cleavage monomer, may be used to reconstitute an active enzyme
dimer. See Wah, et al., 1998, Structure of FokI has implications
for DNA cleavage, PNAS 95:10564-10569; U.S. Pat. No. 5,356,802;
U.S. Pat. No. 5,436,150; U.S. Pat. No. 5,487,994; U.S. Pub.
2005/0064474; U.S. Pub. 2006/0188987; and U.S. Pub. 2008/0131962,
each incorporated by reference.
[0045] Virus targeting using ZFN may include introducing at least
one donor polynucleotide comprising a sequence into the cell. A
donor polynucleotide preferably includes the sequence to be
introduced flanked by an upstream and downstream sequence that
share sequence similarity with either side of the site of
integration in the chromosome. The upstream and downstream
sequences in the donor polynucleotide are selected to promote
recombination between the chromosomal sequence of interest and the
donor polynucleotide. Typically, the donor polynucleotide will be
DNA. The donor polynucleotide may be a DNA plasmid, a bacterial
artificial chromosome (BAC), a yeast artificial chromosome (YAC), a
viral vector, a linear piece of DNA, a PCR fragment, a naked
nucleic acid, and may employ a delivery vehicle such as a liposome.
The sequence of the donor polynucleotide may include exons,
introns, regulatory sequences, or combinations thereof. The double
stranded break is repaired via homologous recombination with the
donor polynucleotide such that the desired sequence is integrated
into the chromosome. In the ZFN-mediated process, a double stranded
break introduced into the target sequence by the ZFN is repaired,
via homologous recombination with the exchange polynucleotide, such
that the sequence in the exchange polynucleotide may be exchanged
with a portion of the target sequence. The presence of the double
stranded break facilitates homologous recombination and repair of
the break. The exchange polynucleotide may be physically integrated
or, alternatively, the exchange polynucleotide may be used as a
template for repair of the break, resulting in the exchange of the
sequence information in the exchange polynucleotide with the
sequence information in that portion of the target sequence. Thus,
a portion of the viral nucleic acid may be converted to the
sequence of the exchange polynucleotide. ZFN methods can include
using a vector to deliver a nucleic acid molecule encoding a ZFN
and, optionally, at least one exchange polynucleotide or at least
one donor polynucleotide to the infected cell.
[0046] Meganucleases are endodeoxyribonucleases characterized by a
large recognition site (double-stranded DNA sequences of 12 to 40
base pairs); as a result this site generally occurs only once in
any given genome. For example, the 18-base pair sequence recognized
by the I-SceI meganuclease would on average require a genome twenty
times the size of the human genome to be found once by chance
(although sequences with a single mismatch occur about three times
per human-sized genome). Meganucleases are therefore considered to
be the most specific naturally occurring restriction enzymes.
Meganucleases can be divided into five families based on sequence
and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box and
PD-(D/E)XK. The most well studied family is that of the LAGLIDADG
proteins, which have been found in all kingdoms of life, generally
encoded within introns or inteins although freestanding members
also exist. The sequence motif, LAGLIDADG, represents an essential
element for enzymatic activity. Some proteins contained only one
such motif, while others contained two; in both cases the motifs
were followed by .about.75-200 amino acid residues having little to
no sequence similarity with other family members. Crystal
structures illustrates mode of sequence specificity and cleavage
mechanism for the LAGLIDADG family: (i) specificity contacts arise
from the burial of extended .beta.-strands into the major groove of
the DNA, with the DNA binding saddle having a pitch and contour
mimicking the helical twist of the DNA; (ii) full hydrogen bonding
potential between the protein and DNA is never fully realized;
(iii) cleavage to generate the characteristic 4-nt 3'-OH overhangs
occurs across the minor groove, wherein the scissile phosphate
bonds are brought closer to the protein catalytic core by a
distortion of the DNA in the central "4-base" region; (iv) cleavage
occurs via a proposed two-metal mechanism, sometimes involving a
unique "metal sharing" paradigm; (v) and finally, additional
affinity and/or specificity contacts can arise from "adapted"
scaffolds, in regions outside the core .alpha./.beta. fold. See
Silva et al., 2011, Meganucleases and other tools for targeted
genome engineering, Curr Gene Ther 11(1):11-27, incorporated by
reference.
[0047] In some embodiments of the invention, a template sequence is
inserted into the genome. In order to introduce nucleotide
modifications to genomic DNA, a DNA repair template containing the
desired sequence must be present during homology directed repair
(HDR). The DNA template is normally transfected into the cell along
with the gRNA/Cas9. The length and binding position of each
homology arm is dependent on the size of the change being
introduced. In the presence of a suitable template, HDR can
introduce significant changes at the Cas9 induced double strand
break.
[0048] Some embodiments of the invention may utilize modified
version of a nuclease. Modified versions of the Cas9 enzyme
containing a single inactive catalytic domain, either RuvC- or
HNH-, are called `nickases`. With only one active nuclease domain,
the Cas9 nickase cuts only one strand of the target DNA, creating a
single-strand break or `nick`. Similar to the inactive dCas9 (RuvC-
and HNH-), a Cas9 nickase is still able to bind DNA based on gRNA
specificity, though nickases will only cut one of the DNA strands.
The majority of CRISPR plasmids are derived from S. pyogenes and
the RuvC domain can be inactivated by a D10A mutation and the HNH
domain can be inactivated by an H840A mutation.
[0049] A single-strand break, or nick, is normally quickly repaired
through the HDR pathway, using the intact complementary DNA strand
as the template. However, two proximal, opposite strand nicks
introduced by a Cas9 nickase are treated as a double strand break,
in what is often referred to as a `double nick` or `dual nickase`
CRISPR system. A double-nick induced double strain break can be
repaired by either NHEJ or HDR depending on the desired effect on
the gene target. At these double strain breaks, insertions and
deletions are caused by the CRISPR/Cas9 complex. In an aspect of
the invention, a deletion is caused by positioning two double
strand breaks proximate to one another, thereby causing a fragment
of the genome to be deleted.
iii. Targeting Moiety
[0050] A nuclease may use the targeting specificity of a guide RNA
(gRNA). As discussed below, guide RNAs or single guide RNAs are
specifically designed to target a virus genome.
[0051] A CRISPR/Cas9 gene editing complex of the invention works
optimally with a guide RNA that targets the viral genome. Guide RNA
(gRNA) (which includes single guide RNA (sgRNA), crisprRNA (crRNA),
transactivating RNA (tracrRNA), any other targeting oligo, or any
combination thereof) leads the CRISPR/Cas9 complex to the viral
genome in order to cause viral genomic disruption. In an aspect of
the invention, CRISPR/Cas9/gRNA complexes are designed to target
specific viruses within a cell. It should be appreciated that any
virus can be targeted using the composition of the invention.
Identification of specific regions of the virus genome aids in
development and designing of CRISPR/Cas9/gRNA complexes.
[0052] In an aspect of the invention, the CRISPR/Cas9/gRNA
complexes are designed to target latent viruses within a cell. Once
transfected within a cell, the CRISPR/Cas9/gRNA complexes cause
repeated insertions or deletions to render the genome
incapacitated, or due to number of insertions or deletions, the
probability of repair is significantly reduced.
[0053] As an example, the Epstein-Barr virus (EBV), also called
human herpesvirus 4 (HHV-4) is inactivated in cells by a
CRISPR/Cas9/gRNA complex of the invention. EBV is a virus of the
herpes family, and is one of the most common viruses in humans. The
virus is approximately 122 nm to 180 nm in diameter and is composed
of a double helix of DNA wrapped in a protein capsid. In this
example, the Raji cell line serves as an appropriate in vitro
model. The Raji cell line is the first continuous human cell line
from hematopoietic origin and cell lines produce an unusual strain
of Epstein-Barr virus while being one of the most extensively
studied EBV models. To target the EBV genomes in the Raji cells, a
CRISPR/Cas9 complex with specificity for EBV is needed. The design
of EBV-targeting CRISPR/Cas9 plasmids consisting of a U6 promoter
driven chimeric guide RNA (sgRNA) and a ubiquitous promoter driven
Cas9 that were obtained from Addgene, Inc. Commercially available
guide RNAs and Cas9 nucleases may be used with the present
invention. An EGFP marker fused after the Cas9 protein allowed
selection of Cas9-positive cells (FIG. 1A).
[0054] In an aspect of the invention, guide RNAs are designed,
whether or not commercially purchased, to target a specific viral
genome. The viral genome is identified and guide RNA to target
selected portions of the viral genome are developed and
incorporated into the composition of the invention. In an aspect of
the invention, a reference genome of a particular strain of the
virus is selected for guide RNA design.
[0055] For example, guide RNAs that target the EBV genome are a
component of the system in the present example. In relation to EBV,
for example, the reference genome from strain B95-8 was used as a
design guide. Within a genome of interest, such as EBV, selected
regions, or genes are targeted. For example, six regions can be
targeted with seven guide RNA designs for different genome editing
purposes (FIG. 1C and Table S1). In relation to EBV, EBNA1 is the
only nuclear Epstein-Barr virus (EBV) protein expressed in both
latent and lytic modes of infection. While EBNA1 is known to play
several important roles in latent infection, EBNA1 is crucial for
many EBV functions including gene regulation and latent genome
replication. Therefore, guide RNAs sgEBV4 and sgEBV5 were selected
to target both ends of the EBNA1 coding region in order to excise
this whole region of the genome. These "structural" targets enable
systematic digestion of the EBV genome into smaller pieces. EBNA3C
and LMP1 are essential for host cell transformation, and guide RNAs
sgEBV3 and sgEBV7 were designed to target the 5' exons of these two
proteins respectively.
iv. Introduce to Cell
[0056] Methods of the invention include introducing into a cell a
nuclease and a sequence-specific targeting moiety. The nuclease is
targeted to viral nucleic acid by means of the sequence-specific
targeting moiety where it then cleaves the viral nucleic acid
without interfering with a host genome. Any suitable method can be
used to deliver the nuclease to the infected cell or tissue. For
example, the nuclease or the gene encoding the nuclease may be
delivered by injection, orally, or by hydrodynamic delivery. The
nuclease or the gene encoding the nuclease may be delivered to
systematic circulation or may be delivered or otherwise localized
to a specific tissue type. The nuclease or gene encoding the
nuclease may be modified or programmed to be active under only
certain conditions such as by using a tissue-specific promoter so
that the encoded nuclease is preferentially or only transcribed in
certain tissue types.
[0057] In some embodiments, specific CRISPR/Cas9/gRNA complexes are
introduced into a cell. A guide RNA is designed to target at least
one category of sequences of the viral genome. In addition to
latent infections this invention can also be used to control
actively replicating viruses by targeting the viral genome before
it is packaged or after it is ejected.
[0058] In some embodiments, a cocktail of guide RNAs may be
introduced into a cell. The guide RNAs are designed to target
numerous categories of sequences of the viral genome. By targeting
several areas along the genome, the double strand break at multiple
locations fragments the genome, lowering the possibility of repair.
Even with repair mechanisms, the large deletions render the virus
incapacitated.
[0059] In some embodiments, several guide RNAs are added to create
a cocktail to target different categories of sequences. For
example, two, five, seven or eleven guide RNAs may be present in a
CRISPR cocktail targeting three different categories of sequences.
However, any number of gRNAs may be introduced into a cocktail to
target categories of sequences. In preferred embodiments, the
categories of sequences are important for genome structure, host
cell transformation, and infection latency, respectively.
[0060] In some aspects of the invention, in vitro experiments allow
for the determination of the most essential targets within a viral
genome. For example, to understand the most essential targets for
effective incapacitation of a genome, subsets of guide RNAs are
transfected into model cells. Assays can determine which guide RNAs
or which cocktail is the most effective at targeting essential
categories of sequences.
[0061] For example, in the case of the EBV genome targeting, seven
guide RNAs in the CRISPR cocktail targeted three different
categories of sequences which are identified as being important for
EBV genome structure, host cell transformation, and infection
latency, respectively. To understand the most essential targets for
effective EBV treatment, Raji cells were transfected with subsets
of guide RNAs. Although sgEBV4/5 reduced the EBV genome by 85%,
they could not suppress cell proliferation as effectively as the
full cocktail (FIG. 3A). Guide RNAs targeting the structural
sequences (sgEBV1/2/6) could stop cell proliferation completely,
despite not eliminating the full EBV load (26% decrease). Given the
high efficiency of genome editing and the proliferation arrest
(FIG. 2), it was suspect that the residual EBV genome signature in
sgEBV1/2/6 was not due to intact genomes but to free-floating DNA
that has been digested out of the EBV genome, i.e. as a false
positive.
[0062] Once CRISPR/Cas9/gRNA complexes are constructed, the
complexes are introduced into a cell. It should be appreciated that
complexes can be introduced into cells in an in vitro model or an
in vivo model. In an aspect of the invention, CRISPR/Cas9/gRNA
complexes are designed to not leave intact genomes of a virus after
transfection and complexes are designed for efficient
transfection.
[0063] Aspects of the invention allow for CRISPR/Cas9/gRNA to be
transfected into cells by various methods, including viral vectors
and non-viral vectors. Viral vectors may include retroviruses,
lentiviruses, adenoviruses, and adeno-associated viruses. It should
be appreciated that any viral vector may be incorporated into the
present invention to effectuate delivery of the CRISPR/Cas9/gRNA
complex into a cell. Some viral vectors may be more effective than
others, depending on the CRISPR/Cas9/gRNA complex designed for
digestion or incapacitation. In an aspect of the invention, the
vectors contain essential components such as origin of replication,
which is necessary for the replication and maintenance of the
vector in the host cell.
[0064] In an aspect of the invention, viral vectors are used as
delivery vectors to deliver the complexes into a cell. Use of viral
vectors as delivery vectors are known in the art. See for example
U.S. Pub. 2009/0017543 to Wilkes et al., the contents of which are
incorporated by reference.
[0065] A retrovirus is a single-stranded RNA virus that stores its
nucleic acid in the form of an mRNA genome (including the 5' cap
and 3' PolyA tail) and targets a host cell as an obligate parasite.
In some methods in the art, retroviruses have been used to
introduce nucleic acids into a cell. Once inside the host cell
cytoplasm the virus uses its own reverse transcriptase enzyme to
produce DNA from its RNA genome, the reverse of the usual pattern,
thus retro (backwards). This new DNA is then incorporated into the
host cell genome by an integrase enzyme, at which point the
retroviral DNA is referred to as a provirus. For example, the
recombinant retroviruses such as the Moloney murine leukemia virus
have the ability to integrate into the host genome in a stable
fashion. They contain a reverse transcriptase that allows
integration into the host genome. Retroviral vectors can either be
replication-competent or replication-defective. In some embodiments
of the invention, retroviruses are incorporated to effectuate
transfection into a cell, however the CRISPR/Cas9/gRNA complexes
are designed to target the viral genome.
[0066] In some embodiments of the invention, lentiviruses, which
are a subclass of retroviruses, are used as viral vectors.
Lentiviruses can be adapted as delivery vehicles (vectors) given
their ability to integrate into the genome of non-dividing cells,
which is the unique feature of lentiviruses as other retroviruses
can infect only dividing cells. The viral genome in the form of RNA
is reverse-transcribed when the virus enters the cell to produce
DNA, which is then inserted into the genome at a random position by
the viral integrase enzyme. The vector, now called a provirus,
remains in the genome and is passed on to the progeny of the cell
when it divides.
[0067] As opposed to lentiviruses, adenoviral DNA does not
integrate into the genome and is not replicated during cell
division. Adenovirus and the related AAV would be potential
approaches as delivery vectors since they do not integrate into the
host's genome. In some aspects of the invention, only the viral
genome to be targeted is effected by the CRISPR/Cas9/gRNA
complexes, and not the host's cells. Adeno-associated virus (AAV)
is a small virus that infects humans and some other primate
species. AAV can infect both dividing and non-dividing cells and
may incorporate its genome into that of the host cell. For example,
because of its potential use as a gene therapy vector, researchers
have created an altered AAV called self-complementary
adeno-associated virus (scAAV). Whereas AAV packages a single
strand of DNA and requires the process of second-strand synthesis,
scAAV packages both strands which anneal together to form double
stranded DNA. By skipping second strand synthesis scAAV allows for
rapid expression in the cell. Otherwise, scAAV carries many
characteristics of its AAV counterpart. Methods of the invention
may incorporate herpesvirus, poxvirus, alphavirus, or vaccinia
virus as a means of delivery vectors.
[0068] In certain embodiments of the invention, non-viral vectors
may be used to effectuate transfection. Methods of non-viral
delivery of nucleic acids include lipofection, nucleofection,
microinjection, biolistics, virosomes, liposomes, immunoliposomes,
polycation or lipid:nucleic acid conjugates, naked DNA, artificial
virions, and agent-enhanced uptake of DNA. Lipofection is described
in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and
lipofection reagents are sold commercially (e.g., Transfectam and
Lipofectin). Cationic and neutral lipids that are suitable for
efficient receptor-recognition lipofection of polynucleotides
include those described in U.S. Pat. No. 7,166,298 to Jessee or
U.S. Pat. No. 6,890,554 to Jesse, the contents of each of which are
incorporated by reference. Delivery can be to cells (e.g. in vitro
or ex vivo administration) or target tissues (e.g. in vivo
administration).
[0069] Synthetic vectors are typically based on cationic lipids or
polymers which can complex with negatively charged nucleic acids to
form particles with a diameter in the order of 100 nm. The complex
protects nucleic acid from degradation by nuclease. Moreover,
cellular and local delivery strategies have to deal with the need
for internalization, release, and distribution in the proper
subcellular compartment. Systemic delivery strategies encounter
additional hurdles, for example, strong interaction of cationic
delivery vehicles with blood components, uptake by the
reticuloendothelial system, kidney filtration, toxicity and
targeting ability of the carriers to the cells of interest.
Modifying the surfaces of the cationic non-virals can minimize
their interaction with blood components, reduce reticuloendothelial
system uptake, decrease their toxicity and increase their binding
affinity with the target cells. Binding of plasma proteins (also
termed opsonization) is the primary mechanism for RES to recognize
the circulating nanoparticles. For example, macrophages, such as
the Kupffer cells in the liver, recognize the opsonized
nanoparticles via the scavenger receptor.
[0070] In some embodiments of the invention, non-viral vectors are
modified to effectuate targeted delivery and transfection.
PEGylation (i.e. modifying the surface with polyethyleneglycol) is
the predominant method used to reduce the opsonization and
aggregation of non-viral vectors and minimize the clearance by
reticuloendothelial system, leading to a prolonged circulation
lifetime after intravenous (i.v.) administration. PEGylated
nanoparticles are therefore often referred as "stealth"
nanoparticles. The nanoparticles that are not rapidly cleared from
the circulation will have a chance to encounter infected cells.
[0071] However, PEG on the surface can decrease the uptake by
target cells and reduce the biological activity. Therefore, to
attach targeting ligand to the distal end of the PEGylated
component is necessary; the ligand is projected beyond the PEG
"shield" to allow binding to receptors on the target cell surface.
When cationic liposome is used as gene carrier, the application of
neutral helper lipid is helpful for the release of nucleic acid,
besides promoting hexagonal phase formation to enable endosomal
escape. In some embodiments of the invention, neutral or anionic
liposomes are developed for systemic delivery of nucleic acids and
obtaining therapeutic effect in experimental animal model.
Designing and synthesizing novel cationic lipids and polymers, and
covalently or noncovalently binding gene with peptides, targeting
ligands, polymers, or environmentally sensitive moieties also
attract many attentions for resolving the problems encountered by
non-viral vectors. The application of inorganic nanoparticles (for
example, metallic nanoparticles, iron oxide, calcium phosphate,
magnesium phosphate, manganese phosphate, double hydroxides, carbon
nanotubes, and quantum dots) in delivery vectors can be prepared
and surface-functionalized in many different ways.
[0072] In some embodiments of the invention, targeted
controlled-release systems responding to the unique environments of
tissues and external stimuli are utilized. Gold nanorods have
strong absorption bands in the near-infrared region, and the
absorbed light energy is then converted into heat by gold nanorods,
the so-called `photothermal effect`. Because the near-infrared
light can penetrate deeply into tissues, the surface of gold
nanorod could be modified with nucleic acids for controlled
release. When the modified gold nanorods are irradiated by
near-infrared light, nucleic acids are released due to
thermo-denaturation induced by the photothermal effect. The amount
of nucleic acids released is dependent upon the power and exposure
time of light irradiation.
[0073] In some embodiments of the invention, liposomes are used to
effectuate transfection into a cell or tissue. The pharmacology of
a liposomal formulation of nucleic acid is largely determined by
the extent to which the nucleic acid is encapsulated inside the
liposome bilayer. Encapsulated nucleic acid is protected from
nuclease degradation, while those merely associated with the
surface of the liposome is not protected. Encapsulated nucleic acid
shares the extended circulation lifetime and biodistribution of the
intact liposome, while those that are surface associated adopt the
pharmacology of naked nucleic acid once they disassociate from the
liposome.
[0074] In some embodiments, the complexes of the invention are
encapsulated in a liposome. Unlike small molecule drugs, nucleic
acids cannot cross intact lipid bilayers, predominantly due to the
large size and hydrophilic nature of the nucleic acid. Therefore,
nucleic acids may be entrapped within liposomes with conventional
passive loading technologies, such as ethanol drop method (as in
SALP), reverse-phase evaporation method, and ethanol dilution
method (as in SNALP).
[0075] In some embodiments, linear polyethylenimine (L-PEI) is used
as a non-viral vector due to its versatility and comparatively high
transfection efficiency. L-PEI has been used to efficiently deliver
genes in vivo into a wide range of organs such as lung, brain,
pancreas, retina, bladder as well as tumor. L-PEI is able to
efficiently condense, stabilize and deliver nucleic acids in vitro
and in vivo.
[0076] Low-intensity ultrasound in combination with microbubbles
has recently acquired much attention as a safe method of gene
delivery. Ultrasound shows tissue-permeabilizing effect. It is
non-invasive and site-specific, and could make it possible to
destroy tumor cells after systemic delivery, while leave
nontargeted organs unaffected. Ultrasound-mediated microbubbles
destruction has been proposed as an innovative method for
noninvasive delivering of drugs and nucleic acids to different
tissues. Microbubbles are used to carry a drug or gene until a
specific area of interest is reached, and then ultrasound is used
to burst the microbubbles, causing site-specific delivery of the
bioactive materials. Furthermore, the ability of albumin-coated
microbubbles to adhere to vascular regions with glycocalix damage
or endothelial dysfunction is another possible mechanism to deliver
drugs even in the absence of ultrasound. See Tsutsui et al., 2004,
The use of microbubbles to target drug delivery, Cardiovasc
Ultrasound 2:23, the contents of which are incorporated by
reference. In ultrasound-triggered drug delivery,
tissue-permeabilizing effect can be potentiated using ultrasound
contrast agents, gas-filled microbubbles. The use of microbubbles
for delivery of nucleic acids is based on the hypothesis that
destruction of DNA-loaded microbubbles by a focused ultrasound beam
during their microvascular transit through the target area will
result in localized transduction upon disruption of the microbubble
shell while sparing non-targeted areas.
[0077] Besides ultrasound-mediated delivery, magnetic targeting
delivery could be used for delivery. Magnetic nanoparticles are
usually entrapped in gene vectors for imaging the delivery of
nucleic acid. Nucleic acid carriers can be responsive to both
ultrasound and magnetic fields, i.e., magnetic and acoustically
active lipospheres (MAALs). The basic premise is that therapeutic
agents are attached to, or encapsulated within, a magnetic micro-
or nanoparticle. These particles may have magnetic cores with a
polymer or metal coating which can be functionalized, or may
consist of porous polymers that contain magnetic nanoparticles
precipitated within the pores. By functionalizing the polymer or
metal coating it is possible to attach, for example, cytotoxic
drugs for targeted chemotherapy or therapeutic DNA to correct a
genetic defect. Once attached, the particle/therapeutic agent
complex is injected into the bloodstream, often using a catheter to
position the injection site near the target. Magnetic fields,
generally from high-field, high-gradient, rare earth magnets are
focused over the target site and the forces on the particles as
they enter the field allow them to be captured and extravasated at
the target.
[0078] Synthetic cationic polymer-based nanoparticles (.about.100
nm diameter) have been developed that offer enhanced transfection
efficiency combined with reduced cytotoxicity, as compared to
traditional liposomes. The incorporation of distinct layers
composed of lipid molecules with varying physical and chemical
characteristics into the polymer nanoparticle formulation resulted
in improved efficiency through better fusion with cell membrane and
entry into the cell, enhanced release of molecules inside the cell,
and reduced intracellular degradation of nanoparticle
complexes.
[0079] In some embodiments, the complexes are conjugated to
nano-systems for systemic therapy, such as liposomes, albumin-based
particles, PEGylated proteins, biodegradable polymer-drug
composites, polymeric micelles, dendrimers, among others. See Davis
et al., 2008, Nanotherapeutic particles: an emerging treatment
modality for cancer, Nat Rev Drug Discov. 7(9):771-782,
incorporated by reference. Long circulating macromolecular carriers
such as liposomes, can exploit the enhanced permeability and
retention effect for preferential extravasation from tumor vessels.
In certain embodiments, the complexes of the invention are
conjugated to or encapsulated into a liposome or polymerosome for
delivery to a cell. For example, liposomal anthracyclines have
achieved highly efficient encapsulation, and include versions with
greatly prolonged circulation such as liposomal daunorubicin and
pegylated liposomal doxorubicin. See Krishna et al.,
Carboxymethylcellulose-sodium based transdermal drug delivery
system for propranolol, J Pharm Pharmacol. 1996 April;
48(4):367-70.
[0080] Liposomal delivery systems provide stable formulation,
provide improved pharmacokinetics, and a degree of `passive` or
`physiological` targeting to tissues. Encapsulation of hydrophilic
and hydrophobic materials, such as potential chemotherapy agents,
are known. See for example U.S. Pat. No. 5,466,468 to Schneider,
which discloses parenterally administrable liposome formulation
comprising synthetic lipids; U.S. Pat. No. 5,580,571, to Hostetler
et al. which discloses nucleoside analogues conjugated to
phospholipids; U.S. Pat. No. 5,626,869 to Nyqvist, which discloses
pharmaceutical compositions wherein the pharmaceutically active
compound is heparin or a fragment thereof contained in a defined
lipid system comprising at least one amphiphatic and polar lipid
component and at least one nonpolar lipid component.
[0081] Liposomes and polymerosomes can contain a plurality of
solutions and compounds. In certain embodiments, the complexes of
the invention are coupled to or encapsulated in polymersomes. As a
class of artificial vesicles, polymersomes are tiny hollow spheres
that enclose a solution, made using amphiphilic synthetic block
copolymers to form the vesicle membrane. Common polymersomes
contain an aqueous solution in their core and are useful for
encapsulating and protecting sensitive molecules, such as drugs,
enzymes, other proteins and peptides, and DNA and RNA fragments.
The polymersome membrane provides a physical barrier that isolates
the encapsulated material from external materials, such as those
found in biological systems. Polymerosomes can be generated from
double emulsions by known techniques, see Lorenceau et al., 2005,
Generation of Polymerosomes from Double-Emulsions, Langmuir
21(20):9183-6, incorporated by reference.
[0082] Some embodiments of the invention provide for a gene gun or
a biolistic particle delivery system. A gene gun is a device for
injecting cells with genetic information, where the payload may be
an elemental particle of a heavy metal coated with plasmid DNA.
This technique may also be referred to as bioballistics or
biolistics. Gene guns have also been used to deliver DNA vaccines.
The gene gun is able to transfect cells with a wide variety of
organic and non-organic species, such as DNA plasmids, fluorescent
proteins, dyes, etc.
[0083] Aspects of the invention provide for numerous uses of
delivery vectors. Selection of the delivery vector is based upon
the cell or tissue targeted and the specific makeup of the
CRISPR/Cas9/gRNA. For example, in the EBV example discussed above,
since lymphocytes are known for being resistant to lipofection,
nucleofection (a combination of electrical parameters generated by
a device called Nucleofector, with cell-type specific reagents to
transfer a substrate directly into the cell nucleus and the
cytoplasm) was necessitated for DNA delivery into the Raji cells.
The Lonza pmax promoter drives Cas9 expression as it offered strong
expression within Raji cells. 24 hours after nucleofection, obvious
EGFP signals were observed from a small proportion of cells through
fluorescent microscopy. The EGFP-positive cell population decreased
dramatically, however, <10% transfection efficiency 48 hours
after nucleofection was measured (FIG. 1B). A CRISPR plasmid that
included the EBV origin of replication sequence, oriP yielded a
transfection efficiency >60% (FIG. 1B).
[0084] Aspects of the invention utilize the CRISPR/Cas9/gRNA
complexes for the targeted delivery. Common known pathways include
transdermal, transmucal, nasal, ocular and pulmonary routes. Drug
delivery systems may include liposomes, proliposomes, microspheres,
gels, prodrugs, cyclodextrins, etc. Aspects of the invention
utilize nanoparticles composed of biodegradable polymers to be
transferred into an aerosol for targeting of specific sites or cell
populations in the lung, providing for the release of the drug in a
predetermined manner and degradation within an acceptable period of
time. Controlled-release technology (CRT), such as transdermal and
transmucosal controlled-release delivery systems, nasal and buccal
aerosol sprays, drug-impregnated lozenges, encapsulated cells, oral
soft gels, iontophoretic devices to administer drugs through skin,
and a variety of programmable, implanted drug-delivery devices are
used in conjunction with the complexes of the invention of
accomplishing targeted and controlled delivery.
v. Cut Viral Nucleic Acid
[0085] Once inside the cell, the CRISPR/Cas9/gRNA complexes target
the viral genome. In an aspect of the invention, the complexes are
targeted to viral genomes. In addition to latent infections this
invention can also be used to control actively replicating viruses
by targeting the viral genome before it is packaged or after it is
ejected. In some embodiments, methods and compositions of the
invention use a nuclease such as Cas9 to target latent viral
genomes, thereby reducing the chances of proliferation. The
nuclease may form a complex with a gRNA (e.g., crRNA+tracrRNA or
sgRNA). The complex cuts the viral nucleic acid in a targeted
fashion to incapacitate the viral genome. As discussed above, the
Cas9 endonuclease causes a double strand break in the viral genome.
By targeted several locations along the viral genome and causing
not a single strand break, but a double strand break, the genome is
effectively cut a several locations along the genome. In a
preferred embodiment, the double strand breaks are designed so that
small deletions are caused, or small fragments are removed from the
genome so that even if natural repair mechanisms join the genome
together, the genome is render incapacitated.
[0086] After introduction into a cell, the CRISPR/Cas9/gRNA
complexes act on the viral genome, genes, transcripts, or other
viral nucleic acid. The double-strand DNA breaks generated by
CRISPR are repaired with small deletions. These deletions will
disrupt the protein coding and hence create knockout effects.
[0087] The nuclease, or a gene encoding the nuclease, may be
delivered into an infected cell by transfection. For example, the
infected cell can be transfected with DNA that encodes Cas9 and
gRNA (on a single piece or separate pieces). The gRNAs are designed
to localize the Cas9 endonuclease at one or several locations along
the viral genome. The Cas9 endonuclease causes double strand breaks
in the genome, causing small fragments to be deleted from the viral
genome. Even with repair mechanisms, the deletions render the viral
genome incapacitated.
vi. Host Genome
[0088] It will be appreciated that method and compositions of the
invention can be used to target viral nucleic acid without
interfering with host genetic material. Methods and compositions of
the invention employ a targeting moiety such as a guide RNA that
has a sequence that hybridizes to a target within the viral
sequence. Methods and compositions of the invention may further use
a targeted nuclease such as the cas9 enzyme, or a vector encoding
such a nuclease, which uses the gRNA to bind exclusively to the
viral genome and make double stranded cuts, thereby removing the
viral sequence from the host.
[0089] Where the targeting moiety includes a guide RNA, the
sequence for the gRNA, or the guide sequence, can be determined by
examination of the viral sequence to find regions of about 20
nucleotides that are adjacent to a protospacer adjacent motif (PAM)
and that do not also appear in the host genome adjacent to the
protospacer motif.
[0090] Preferably a guide sequence that satisfies certain
similarity criteria (e.g., at least 60% identical with identity
biased toward regions closer to the PAM) so that a gRNA/cas9
complex made according to the guide sequence will bind to and
digest specified features or targets in the viral sequence without
interfering with the host genome. Preferably, the guide RNA
corresponds to a nucleotide string next to a protospacer adjacent
motif (PAM) (e.g., NGG, where N is any nucleotide) in the viral
sequence. Preferably, the host genome lacks any region that (1)
matches the nucleotide string according to a predetermined
similarity criteria and (2) is also adjacent to the PAM. The
predetermined similarity criteria may include, for example, a
requirement of at least 12 matching nucleotides within 20
nucleotides 5' to the PAM and may also include a requirement of at
least 7 matching nucleotides within 10 nucleotides 5' to the PAM.
An annotated viral genome (e.g., from GenBank) may be used to
identify features of the viral sequence and finding the nucleotide
string next to a protospacer adjacent motif (PAM) in the viral
sequence within a selected feature (e.g., a viral replication
origin, a terminal repeat, a replication factor binding site, a
promoter, a coding sequence, or a repetitive region) of the viral
sequence. The viral sequence and the annotations may be obtained
from a genome database.
[0091] Where multiple candidate gRNA targets are found in the viral
genome, selection of the sequence to be the template for the guide
RNA may favor the candidate target closest to, or at the 5' most
end of, a targeted feature as the guide sequence. The selection may
preferentially favor sequences with neutral (e.g., 40% to 60%) GC
content. Additional background regarding the RNA-directed targeting
by endonuclease is discussed in U.S. Pub. 2015/0050699; U.S. Pub.
20140356958; U.S. Pub. 2014/0349400; U.S. Pub. 2014/0342457; U.S.
Pub. 2014/0295556; and U.S. Pub. 2014/0273037, the contents of each
of which are incorporated by reference for all purposes. Due to the
existence of human genomes background in the infected cells, a set
of steps are provided to ensure high efficiency against the viral
genome and low off-target effect on the human genome. Those steps
may include (1) target selection within viral genome, (2) avoiding
PAM+target sequence in host genome, (3) methodologically selecting
viral target that is conserved across strains, (4) selecting target
with appropriate GC content, (5) control of nuclease expression in
cells, (6) vector design, (7) validation assay, others and various
combinations thereof. A targeting moiety (such as a guide RNA)
preferably binds to targets within certain categories such as (i)
latency related targets, (ii) infection and symptom related
targets, and (iii) structure related targets.
[0092] A first category of targets for gRNA includes
latency-related targets. The viral genome requires certain features
in order to maintain the latency. These features include, but not
limited to, master transcription regulators, latency-specific
promoters, signaling proteins communicating with the host cells,
etc. If the host cells are dividing during latency, the viral
genome requires a replication system to maintain genome copy level.
Viral replication origin, terminal repeats, and replication factors
binding to the replication origin are great targets. Once the
functions of these features are disrupted, the viruses may
reactivate, which can be treated by conventional antiviral
therapies.
[0093] A second category of targets for gRNA includes
infection-related and symptom-related targets. Virus produces
various molecules to facilitate infection. Once gained entrance to
the host cells, the virus may start lytic cycle, which can cause
cell death and tissue damage (HBV). In certain cases, such as
HPV16, cell products (E6 and E7 proteins) can transform the host
cells and cause cancers. Disrupting the key genome sequences
(promoters, coding sequences, etc) producing these molecules can
prevent further infection, and/or relieve symptoms, if not curing
the disease.
[0094] A third category of targets for gRNA includes
structure-related targets. Viral genome may contain repetitive
regions to support genome integration, replication, or other
functions. Targeting repetitive regions can break the viral genome
into multiple pieces, which physically destroys the genome.
[0095] Where the nuclease is a cas protein, the targeting moiety is
a guide RNA. Each cas protein requires a specific PAM next to the
targeted sequence (not in the guide RNA). This is the same as for
human genome editing. The current understanding the guide
RNA/nuclease complex binds to PAM first, then searches for homology
between guide RNA and target genome. Sternberg et al., 2014, DNA
interrogation by the CRISPR RNA-guided endonuclease Cas9, Nature
507(7490):62-67. Once recognized, the DNA is digested 3-nt upstream
of PAM. These results suggest that off-target digestion requires
PAM in the host DNA, as well as high affinity between guide RNA and
host genome right before PAM.
[0096] It may be preferable to use a targeting moiety that targets
portions of the viral genome that are highly conserved. Viral
genomes are much more variable than human genomes. In order to
target different strains, the guide RNA will preferably target
conserved regions. As PAM is important to initial sequence
recognition, it is also essential to have PAM in the conserved
region.
[0097] In a preferred embodiment, methods of the invention are used
to deliver a nucleic acid to cells. The nucleic acid delivered to
the cells may include a gRNA having the determined guide sequence
or the nucleic acid may include a vector, such as a plasmid, that
encodes an enzyme that will act against the target genetic
material. Expression of that enzyme allows it to degrade or
otherwise interfere with the target genetic material. The enzyme
may be a nuclease such as the Cas9 endonuclease and the nucleic
acid may also encode one or more gRNA having the determined guide
sequence.
[0098] The gRNA targets the nuclease to the target genetic
material. Where the target genetic material includes the genome of
a virus, gRNAs complementary to parts of that genome can guide the
degredation of that genome by the nuclease, thereby preventing any
further replication or even removing any intact viral genome from
the cells entirely. By these means, latent viral infections can be
targeted for eradication.
[0099] The host cells may grow at different rate, based on the
specific cell type. High nuclease expression is necessary for fast
replicating cells, whereas low expression help avoiding off-target
cutting in non-infected cells. Control of nuclease expression can
be achieved through several aspects. If the nuclease is expressed
from a vector, having the viral replication origin in the vector
can increase the vector copy number dramatically, only in the
infected cells. Each promoter has different activities in different
tissues. Gene transcription can be tuned by choosing different
promoters. Transcript and protein stability can also be tuned by
incorporating stabilizing or destabilizing (ubiquitin targeting
sequence, etc) motif into the sequence.
[0100] Specific promoters may be used for the gRNA sequence, the
nuclease (e.g., cas9), other elements, or combinations thereof. For
example, in some embodiments, the gRNA is driven by a U6 promoter.
A vector may be designed that includes a promoter for protein
expression (e.g., using a promoter as described in the vector sold
under the trademark PMAXCLONING by Lonza Group Ltd (Basel,
Switzerland). A vector may be a plasmid (e.g., created by synthesis
instrument 255 and recombinant DNA lab equipment). In certain
embodiments, the plasmid includes a U6 promoter driven gRNA or
chimeric guide RNA (sgRNA) and a ubiquitous promoter-driven cas9.
Optionally, the vector may include a marker such as EGFP fused
after the cas9 protein to allow for later selection of cas9+ cells.
It is recognized that cas9 can use a gRNA (similar to the CRISPR
RNA (crRNA) of the original bacterial system) with a complementary
trans-activating crRNA (tracrRNA) to target viral sequences
complementary to the gRNA. It has also been shown that cas9 can be
programmed with a single RNA molecule, a chimera of the gRNA and
tracrRNA. The single guide RNA (sgRNA) can be encoded in a plasmid
and transcription of the sgRNA can provide the programming of cas9
and the function of the tracrRNA. See Jinek, 2012, A programmable
dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,
Science 337:816-821 and especially FIG. 5A therein for
background.
[0101] Using the above principles, methods and compositions of the
invention may be used to target viral nucleic acid in an infected
host without adversely influencing the host genome.
[0102] For additional background see Hsu, 2013, DNA targeting
specificity of RNA-guided Cas9 nucleases, Nature Biotechnology
31(9):827-832; and Jinek, 2012, A programmable dual-RNA-guided DNA
endonuclease in adaptive bacterial immunity, Science 337:816-821,
the contents of each of which are incorporated by reference. Since
the targeted locations are selected to be within certain categories
such as (i) latency related targets, (ii) infection and symptom
related targets, or (iii) structure related targets, cleavage of
those sequences inactivates the virus and removes it from the host.
Since the targeting RNA (the gRNA or sgRNA) is designed to satisfy
according to similarity criteria that matches the target in the
viral genetic sequence without any off-target matching the host
genome, the latent viral genetic material is removed from the host
without any interference with the host genome.
INCORPORATION BY REFERENCE
[0103] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in
their entirety for all purposes.
EQUIVALENTS
[0104] Various modifications of the invention and many further
embodiments thereof, in addition to those shown and described
herein, will become apparent to those skilled in the art from the
full contents of this document, including references to the
scientific and patent literature cited herein. The subject matter
herein contains important information, exemplification and guidance
that can be adapted to the practice of this invention in its
various embodiments and equivalents thereof.
EXAMPLES
Example 1
Targeting EBV
[0105] Burkitt's lymphoma cell lines Raji, Namalwa, and DG-75 were
obtained from ATCC and cultured in RPMI 1640 supplemented with 10%
FBS and PSA, following ATCC recommendation. Human primary lung
fibroblast IMR-90 was obtained from Coriell and cultured in
Advanced DMEM/F-12 supplemented with 10% FBS and PSA.
[0106] Plasmids consisting of a U6 promoter driven chimeric guide
RNA (sgRNA) and a ubiquitous promoter driven Cas9 were obtained
from addgene, as described by Cong L et al. (2013) Multiplex Genome
Engineering Using CRISPR/Cas Systems. Science 339:819-823. An EGFP
marker fused after the Cas9 protein allowed selection of
Cas9-positive cells (FIG. 1A). We adapted a modified chimeric guide
RNA design for more efficient Pol-III transcription and more stable
stem-loop structure (Chen B et al. (2013) Dynamic Imaging of
Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas
System. Cell 155:1479-1491).
[0107] We obtained pX458 from Addgene, Inc. A modified CMV promoter
with a synthetic intron (pmax) was PCR amplified from Lonza control
plasmid pmax-GFP. A modified guide RNA sgRNA(F+E) was ordered from
IDT. EBV replication origin oriP was PCR amplified from B95-8
transformed lymphoblastoid cell line GM12891. We used standard
cloning protocols to clone pmax, sgRNA(F+E) and oriP to pX458, to
replace the original CAG promoter, sgRNA and f1 origin. We designed
EBV sgRNA based on the B95-8 reference, and ordered DNA oligos from
IDT. The original sgRNA place holder in pX458 serves as the
negative control.
[0108] Lymphocytes are known for being resistant to lipofection,
and therefore we used nucleofection for DNA delivery into Raji
cells. We chose the Lonza pmax promoter to drive Cas9 expression as
it offered strong expression within Raji cells. We used the Lonza
Nucleofector II for DNA delivery. 5 million Raji or DG-75 cells
were transfected with 5 ug plasmids in each 100-ul reaction. Cell
line Kit V and program M-013 were used following Lonza
recommendation. For IMR-90, 1 million cells were transfected with 5
ug plasmids in 100 ul Solution V, with program T-030 or X-005. 24
hours after nucleofection, we observed obvious EGFP signals from a
small proportion of cells through fluorescent microscopy. The
EGFP-positive cell population decreased dramatically after that,
however, and we measured <10% transfection efficiency 48 hours
after nucleofection (FIG. 1B). We attributed this transfection
efficiency decrease to the plasmid dilution with cell division. To
actively maintain the plasmid level within the host cells, we
redesigned the CRISPR plasmid to include the EBV origin of
replication sequence, oriP. With active plasmid replication inside
the cells, the transfection efficiency rose to >60% (FIG.
1B).
[0109] To design guide RNA targeting the EBV genome, we relied on
the EBV reference genome from strain B95-8. We targeted six regions
with seven guide RNA designs for different genome editing purposes
(FIG. 1C and Table S1).
TABLE-US-00001 TABLE S1 Guide RNA target sequences sgEBV1 (SEQ ID
NO: 1) GCCCTGGACCAACCCGGCCC sgEBV2 (SEQ ID NO: 2)
GGCCGCTGCCCCGCTCCGGG sgEBB3 (SEQ ID NO: 3) GGAAGACAATGTGCCGCCA
sgEBV4 (SEQ ID NO: 4) TCTGGACCAGAAGGCTCCGG sgEBV5 (SEQ ID NO: 5)
GCTGCCGCGGAGGGTGATGA sgEBV6 (SEQ ID NO: 6) GGTGGCCCACCGGGTCCGCT
sgEBV7 (SEQ ID NO: 7) GTCCTCGAGGGGGCCGTCGC
[0110] EBNA1 is crucial for many EBV functions including gene
regulation and latent genome replication. We targeted guide RNA
sgEBV4 and sgEBV5 to both ends of the EBNA1 coding region in order
to excise this whole region of the genome. Guide RNAs sgEBV1, 2 and
6 fall in repeat regions, so that the success rate of at least one
CRISPR cut is multiplied. These "structural" targets enable
systematic digestion of the EBV genome into smaller pieces. EBNA3C
and LMP1 are essential for host cell transformation, and we
designed guide RNAs sgEBV3 and sgEBV7 to target the 5' exons of
these two proteins respectively.
[0111] EBV Genome Editing. The double-strand DNA breaks generated
by CRISPR are repaired with small deletions. These deletions will
disrupt the protein coding and hence create knockout effects.
SURVEYOR assays confirmed efficient editing of individual sites
(FIG. 5). Beyond the independent small deletions induced by each
guide RNA, large deletions between targeting sites can
systematically destroy the EBV genome. Guide RNA sgEBV2 targets a
region with twelve 125-bp repeat units (FIG. 2A). PCR amplicon of
the whole repeat region gave a .about.1.8-kb band (FIG. 2B). After
5 or 7 days of sgEBV2 transfection, we obtained .about.0.4-kb bands
from the same PCR amplification (FIG. 2B). The .about.1.4-kb
deletion is the expected product of repair ligation between cuts in
the first and the last repeat unit (FIG. 2A).
[0112] DNA sequences flanking sgRNA targets were PCR amplified with
Phusion DNA polymerase. SURVEYOR assays were performed following
manufacturer's instruction. DNA amplicons with large deletions were
TOPO cloned and single colonies were used for Sanger sequencing.
EBV load was measured with Taqman digital PCR on Fluidigm BioMark.
A Taqman assay targeting a conserved human locus was used for human
DNA normalization. 1 ng of single-cell whole-genome amplification
products from Fluidigm C1 were used for EBV quantitative PCR.
[0113] We further demonstrated that it is possible to delete
regions between unique targets (FIG. 2C). Six days after sgEBV4-5
transfection, PCR amplification of the whole flanking region (with
primers EBV4F and 5R) returned a shorter amplicon, together with a
much fainter band of the expected 2 kb (FIG. 2D). Sanger sequencing
of amplicon clones confirmed the direct connection of the two
expected cutting sites (FIG. 2F). A similar experiment with
sgEBV3-5 also returned an even larger deletion, from EBNA3C to
EBNA1 (FIG. 2D-E).
[0114] Additional information such as primer design is shown in
Wang and Quake, 2014, RNA-guided endonuclease provides a
therapeutic strategy to cure latent herpesviridae infection, PNAS
111(36):13157-13162 and in the Supporting Information to that
article published online at the PNAS website, and the contents of
both of those documents are incorporated by reference for all
purposes.
[0115] Cell Proliferation Arrest With EBV Genome Destruction. Two
days after CRISPR transfection, we flow sorted EGFP-positive cells
for further culture and counted the live cells daily. As expected,
cells treated with Cas9 plasmids which lacked oriP or sgEBV lost
EGFP expression within a few days and proliferated with a rate
similar rate to the untreated control group (FIG. 3A). Plasmids
with Cas9-oriP and a scrambled guide RNA maintained EGFP expression
after 8 days, but did not reduce the cell proliferation rate.
Treatment with the mixed cocktail sgEBV1-7 resulted in no
measurable cell proliferation and the total cell count either
remained constant or decreased (FIG. 3A). Flow cytometry scattering
signals clearly revealed alterations in the cell morphology after
sgEBV1-7 treatment, as the majority of the cells shrank in size
with increasing granulation (FIG. 3B-D, population P4 to P3 shift).
Cells in population P3 also demonstrated compromised membrane
permeability by DAPI staining (FIG. 3E-G). To rule out the
possibility of CRISPR cytotoxicity, especially with multiple guide
RNAs, we performed the same treatment on two other samples: the
EBV-negative Burkitt's lymphoma cell line DG-75 (FIG. 6) and
primary human lung fibroblast IMR90 (FIG. 7). Eight and nine days
after transfection the cell proliferation rates did not change from
the untreated control groups, suggesting neglectable
cytotoxicity.
[0116] Previous studies have attributed the EBV tumorigenic ability
to its interruption of host cell apoptosis (Ruf I K et al. (1999)
Epstein-Barr Virus Regulates c-MYC, Apoptosis, and Tumorigenicity
in Burkitt Lymphoma. Molecular and Cellular Biology 19:1651-1660).
Suppressing EBV activities may therefore restore the apoptosis
process, which could explain the cell death observed in our
experiment Annexin V staining revealed a distinct subpopulation of
cells with intact cell membrane but exposed phosphatidylserine,
suggesting cell death through apoptosis (FIG. 3E-G). Bright field
microscopy showed obvious apoptotic cell morphology (FIG. 3H-I) and
fluorescent staining demonstrated drastic DNA fragmentation (FIG.
3J-M). Altogether this evidence suggests restoration of the normal
host cell apoptosis pathway after EBV genome destruction.
[0117] Complete Clearance Of EBV In A Subpopulation. To study the
potential connection between cell proliferation arrest and EBV
genome editing, we quantified the EBV load in different samples
with digital PCR targeting EBNA1. Another Taqman assay targeting a
conserved human somatic locus served as the internal control for
human DNA normalization. On average, each untreated Raji cell has
42 copies of EBV genome (FIG. 4A). Cells treated with a Cas9
plasmid that lacked oriP or sgEBV did not have an obvious
difference in EBV load difference from the untreated control. Cells
treated with a Cas9-plasmid with oriP but no sgEBV had an EBV load
that was reduced by .about.50%. In conjunction with the prior
observation that cells from this experiment did not show any
difference in proliferation rate, we interpret this as likely due
to competition for EBNA1 binding during plasmid replication. The
addition of the guide RNA cocktail sgEBV1-7 to the transfection
dramatically reduced the EBV load. Both the live and dead cells
have >60% EBV decrease comparing to the untreated control.
[0118] Although we provided seven guide RNAs at the same molar
ratio, the plasmid transfection and replication process is likely
quite stochastic. Some cells will inevitably receive different
subsets or mixtures of the guide RNA cocktail, which might affect
the treatment efficiency. To control for such effects, we measured
EBV load at the single cell level by employing single-cell
whole-genome amplification with an automated microfluidic system.
We loaded freshly cultured Raji cells onto the microfluidic chip
and captured 81 single cells (FIG. 4B). For the sgEBV1-7 treated
cells, we flow sorted the live cells eight days after transfection
and captured 91 single cells (FIG. 4C). Following manufacturer's
instruction, we obtained .about.150 ng amplified DNA from each
single cell reaction chamber. For quality control purposes we
performed 4-loci human somatic DNA quantitative PCR on each single
cell amplification product (Wang J, Fan H C, Behr B, Quake S R
(2012) Genome-wide single-cell analysis of recombination activity
and de novo mutation rates in human sperm. Cell 150:402-412) and
required positive amplification from at least one locus. 69
untreated single-cell products passed the quality control and
displayed a log-normal distribution of EBV load (FIG. 4D) with
almost every cell displaying significant amounts of EBV genomic
DNA. We calibrated the quantitative PCR assay with a subclone of
Namalwa Burkitt's lymphoma cells, which contain a single integrated
EBV genome. The single-copy EBV measurements gave a Ct of 29.8,
which enabled us to determine that the mean Ct of the 69 Raji
single cell samples corresponded to 42 EBV copies per cells, in
concordance with the bulk digital PCR measurement. For the sgEBV1-7
treated sample, 71 single-cell products passed the quality control
and the EBV load distribution was dramatically wider (FIG. 4E).
While 22 cells had the same EBV load as the untreated cells, 19
cells had no detectable EBV and the remaining 30 cells displayed
dramatic EBV load decrease from the untreated sample.
[0119] Essential Targets For EBV Treatment. The seven guide RNAs in
our CRISPR cocktail target three different categories of sequences
which are important for EBV genome structure, host cell
transformation, and infection latency, respectively. To understand
the most essential targets for effective EBV treatment, we
transfected Raji cells with subsets of guide RNAs. Although
sgEBV4/5 reduced the EBV genome by 85%, they could not suppress
cell proliferation as effectively as the full cocktail (FIG. 3A).
Guide RNAs targeting the structural sequences (sgEBV1/2/6) could
stop cell proliferation completely, despite not eliminating the
full EBV load (26% decrease). Given the high efficiency of genome
editing and the proliferation arrest (FIG. 2), we suspect that the
residual EBV genome signature in sgEBV1/2/6 was not due to intact
genomes but to free-floating DNA that has been digested out of the
EBV genome, i.e. as a false positive. We conclude that systematic
destruction of EBV genome structure appears to be more effective
than targeting specific key proteins for EBV treatment.
Example 2
Targeting Hepatitis B Virus (HBV)
[0120] Methods and materials of the present invention may be used
to apply targeted endonuclease to specific genetic material such as
a latent viral genome like the hepatitis B virus (HBV). The
invention further provides for the efficient and safe delivery of
nucleic acid (such as a DNA plasmid) into target cells (e.g.,
hepatocytes). In one embodiment, methods of the invention use
hydrodynamic gene delivery to target HBV.
[0121] FIG. 10 diagrams the HBV genome. It may be preferable to
receive annotations for the HBV genome (i.e., that identify
important features of the genome) and choose a candidate for
targeting by enzymatic degredation that lies within one of those
features, such as a viral replication origin, a terminal repeat, a
replication factor binding site, a promoter, a coding sequence, and
a repetitive region.
[0122] HBV, which is the prototype member of the family
Hepadnaviridae, is a 42 nm partially double stranded DNA virus,
composed of a 27 nm nucleocapsid core (HBcAg), surrounded by an
outer lipoprotein coat (also called envelope) containing the
surface antigen (HBsAg). The virus includes an enveloped virion
containing 3 to 3.3 kb of relaxed circular, partially duplex DNA
and virion-associated DNA-dependent polymerases that can repair the
gap in the virion DNA template and has reverse transcriptase
activities. HBV is a circular, partially double-stranded DNA virus
of approximately 3200 bp with four overlapping ORFs encoding the
polymerase (P), core (C), surface (S) and X proteins. In infection,
viral nucleocapsids enter the cell and reach the nucleus, where the
viral genome is delivered. In the nucleus, second-strand DNA
synthesis is completed and the gaps in both strands are repaired to
yield a covalently closed circular DNA molecule that serves as a
template for transcription of four viral RNAs that are 3.5, 2.4,
2.1, and 0.7 kb long. These transcripts are polyadenylated and
transported to the cytoplasm, where they are translated into the
viral nucleocapsid and precore antigen (C, pre-C), polymerase (P),
envelope L (large), M (medium), S (small)), and transcriptional
transactivating proteins (X). The envelope proteins insert
themselves as integral membrane proteins into the lipid membrane of
the endoplasmic reticulum (ER). The 3.5 kb species, spanning the
entire genome and termed pregenomic RNA (pgRNA), is packaged
together with HBV polymerase and a protein kinase into core
particles where it serves as a template for reverse transcription
of negative-strand DNA. The RNA to DNA conversion takes place
inside the particles.
[0123] Numbering of basepairs on the HBV genome is based on the
cleavage site for the restriction enzyme EcoR1 or at homologous
sites, if the EcoR1 site is absent. However, other methods of
numbering are also used, based on the start codon of the core
protein or on the first base of the RNA pregenome. Every base pair
in the HBV genome is involved in encoding at least one of the HBV
protein. However, the genome also contains genetic elements which
regulate levels of transcription, determine the site of
polyadenylation, and even mark a specific transcript for
encapsidation into the nucleocapsid. The four ORFs lead to the
transcription and translation of seven different HBV proteins
through use of varying in-frame start codons. For example, the
small hepatitis B surface protein is generated when a ribosome
begins translation at the ATG at position 155 of the adw genome.
The middle hepatitis B surface protein is generated when a ribosome
begins at an upstream ATG at position 3211, resulting in the
addition of 55 amino acids onto the 5' end of the protein.
[0124] ORF P occupies the majority of the genome and encodes for
the hepatitis B polymerase protein. ORF S encodes the three surface
proteins. ORF C encodes both the hepatitis e and core protein. ORF
X encodes the hepatitis B X protein. The HBV genome contains many
important promoter and signal regions necessary for viral
replication to occur. The four ORFs transcription are controlled by
four promoter elements (preS1, preS2, core and X), and two enhancer
elements (Enh I and Enh II). All HBV transcripts share a common
adenylation signal located in the region spanning 1916-1921 in the
genome. Resulting transcripts range from 3.5 nucleotides to 0.9
nucleotides in length. Due to the location of the core/pregenomic
promoter, the polyadenylation site is differentially utilized. The
polyadenylation site is a hexanucleotide sequence (TATAAA) as
opposed to the canonical eukaryotic polyadenylation signal sequence
(AATAAA). The TATAAA is known to work inefficiently (9), suitable
for differential use by HBV.
[0125] There are four known genes encoded by the genome, called C,
X, P, and S. The core protein is coded for by gene C (HBcAg), and
its start codon is preceded by an upstream in-frame AUG start codon
from which the pre-core protein is produced. HBeAg is produced by
proteolytic processing of the pre-core protein. The DNA polymerase
is encoded by gene P. Gene S is the gene that codes for the surface
antigen (HBsAg). The HBsAg gene is one long open reading frame but
contains three in-frame start (ATG) codons that divide the gene
into three sections, pre-S1, pre-S2, and S. Because of the multiple
start codons, polypeptides of three different sizes called large,
middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S) are produced.
The function of the protein coded for by gene X is not fully
understood but it is associated with the development of liver
cancer. It stimulates genes that promote cell growth and
inactivates growth regulating molecules.
[0126] With reference to FIG. 10, HBV starts its infection cycle by
binding to the host cells with PreS1. Guide RNA against PreS1
locates at the 5' end of the coding sequence. Endonuclease
digestion will introduce insertion/deletion, which leads to frame
shift of PreS1 translation. HBV replicates its genome through the
form of long RNA, with identical repeats DR1 and DR2 at both ends,
and RNA encapsidation signal epsilon at the 5' end. The reverse
transcriptase domain (RT) of the polymerase gene converts the RNA
into DNA. Hbx protein is a key regulator of viral replication, as
well as host cell functions. Digestion guided by RNA against RT
will introduce insertion/deletion, which leads to frame shift of RT
translation. Guide RNAs sgHbx and sgCore can not only lead to frame
shift in the coding of Hbx and HBV core protein, but also deletion
the whole region containing DR2-DR1-Epsilon. The four sgRNA in
combination can also lead to systemic destruction of HBV genome
into small pieces.
[0127] HBV replicates its genome by reverse transcription of an RNA
intermediate. The RNA templates is first converted into
single-stranded DNA species (minus-strand DNA), which is
subsequently used as templates for plus-strand DNA synthesis. DNA
synthesis in HBV use RNA primers for plus-strand DNA synthesis,
which predominantly initiate at internal locations on the
single-stranded DNA. The primer is generated via an RNase H
cleavage that is a sequence independent measurement from the 5' end
of the RNA template. This 18 nt RNA primer is annealed to the 3'
end of the minus-strand DNA with the 3' end of the primer located
within the 12 nt direct repeat, DR1. The majority of plus-strand
DNA synthesis initiates from the 12 nt direct repeat, DR2, located
near the other end of the minus-strand DNA as a result of primer
translocation. The site of plus-strand priming has consequences. In
situ priming results in a duplex linear (DL) DNA genome, whereas
priming from DR2 can lead to the synthesis of a relaxed circular
(RC) DNA genome following completion of a second template switch
termed circularization. It remains unclear why hepadnaviruses have
this added complexity for priming plus-strand DNA synthesis, but
the mechanism of primer translocation is a potential therapeutic
target. As viral replication is necessary for maintenance of the
hepadnavirus (including the human pathogen, hepatitis B virus)
chronic carrier state, understanding replication and uncovering
therapeutic targets is critical for limiting disease in
carriers.
[0128] In some embodiments, systems and methods of the invention
target the HBV genome by finding a nucleotide string within a
feature such as PreS1.
[0129] Guide RNA against PreS1 locates at the 5' end of the coding
sequence. Thus it is a good candidate for targeting because it
represents one of the 5'-most targets in the coding sequence.
Endonuclease digestion will introduce insertion/deletion, which
leads to frame shift of PreS1 translation. HBV replicates its
genome through the form of long RNA, with identical repeats DR1 and
DR2 at both ends, and RNA encapsidation signal epsilon at the 5'
end.
[0130] The reverse transcriptase domain (RT) of the polymerase gene
converts the RNA into DNA. Hbx protein is a key regulator of viral
replication, as well as host cell functions. Digestion guided by
RNA against RT will introduce insertion/deletion, which leads to
frame shift of RT translation.
[0131] Guide RNAs sgHbx and sgCore can not only lead to frame shift
in the coding of Hbx and HBV core protein, but also deletion the
whole region containing DR2-DR1-Epsilon. The four sgRNA in
combination can also lead to systemic destruction of HBV genome
into small pieces. In some embodiments, method of the invention
include creating one or several guide RNAs against key features
within a genome such as the HBV genome shown in FIG. 10.
[0132] FIG. 10 shows key parts in the HBV genome targeted by CRISPR
guide RNAs. To achieve the CRISPR activity in cells, expression
plasmids coding cas9 and guide RNAs are delivered to cells of
interest (e.g., cells carrying HBV DNA). To demonstrate in an in
vitro assay, anti-HBV effect may be evaluated by monitoring cell
proliferation, growth, and morphology as well as analyzing DNA
integrity and HBV DNA load in the cells.
[0133] The described method may be validated using an in vitro
assay. To demonstrate, an in vitro assay is performed with cas9
protein and DNA amplicons flanking the target regions. Here, the
target is amplified and the amplicons are incubated with cas9 and a
gRNA having the selected nucleotide sequence for targeting. As
shown in FIG. 11, DNA electrophoresis shows strong digestion at the
target sites.
[0134] FIG. 11 shows a gel resulting from an in vitro CRISPR assay
against HBV. Lanes 1, 3, and 6: PCR amplicons of HBV genome
flanking RT, Hbx-Core, and PreS1. Lane 2, 4, 5, and 7: PCR
amplicons treated with sgHBV-RT, sgHBV-Hbx, sgHBV-Core,
sgHBV-PreS1. The presence of multiple fragments especially visible
in lanes 5 and 7 show that sgHBV-Core and sgHBV-PreS1 provide
especially attractive targets in the context of HBV and that use of
systems and methods of the invention may be shown to be effective
by an in vitro validation assay.
Sequence CWU 1
1
7120DNAArtificial SequenceSynthetic sgEBV1 guide RNA targeting
Sequence 1gccctggacc aacccggccc 20220DNAArtificial
SequenceSynthetic sgEBV2 guide RNA targeting Sequence 2ggccgctgcc
ccgctccggg 20319DNAArtificial SequenceSynthetic sgEBV3 guide RNA
targeting Sequence 3ggaagacaat gtgccgcca 19420DNAArtificial
SequenceSynthetic sgEBV4 guide RNA targeting Sequence 4tctggaccag
aaggctccgg 20520DNAArtificial SequenceSynthetic sgEBV5 guide RNA
targeting Sequence 5gctgccgcgg agggtgatga 20620DNAArtificial
SequenceSynthetic sgEBV6 guide RNA targeting Sequence 6ggtggcccac
cgggtccgct 20720DNAArtificial SequenceSynthetic sgEBV7 guide RNA
targeting Sequence 7gtcctcgagg gggccgtcgc 20
* * * * *