U.S. patent application number 15/362519 was filed with the patent office on 2017-06-29 for compositions and methods of delivering treatments for 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 | 20170182190 15/362519 |
Document ID | / |
Family ID | 53366336 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170182190 |
Kind Code |
A1 |
Quake; Stephen R. ; et
al. |
June 29, 2017 |
Compositions and Methods of Delivering Treatments for Latent Viral
Infections
Abstract
Viral infection is a persistent cause of human disease. Guided
nuclease systems of the invention target the genomes of viral
infections, rendering the viruses incapacitated. The invention
further provides delivery methods and compositions for antiviral
therapeutics. Methods and compositions are provided for targeted
delivery of antiviral therapeutics into cells of interest using,
for example, viral vectors such as adenovirus, AAV, and replication
incompetent HSV. These and other delivery systems can be used as
vehicles to deliver DNA vectors encoding a nuclease or a
cell-killing gene. These delivery methods can also be used to
deliver naked DNA or RNA, protein products, plasmids containing a
promoter that is active only in a latent viral state which drives a
cell-killing gene, or other therapeutic agents.
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 |
Stanford |
CA |
US |
|
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University
Stanford
CA
|
Family ID: |
53366336 |
Appl. No.: |
15/362519 |
Filed: |
November 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2015/033199 |
May 29, 2015 |
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15362519 |
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62029072 |
Jul 25, 2014 |
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62005395 |
May 30, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1133 20130101;
Y02A 50/465 20180101; Y02A 50/393 20180101; A61P 31/14 20180101;
Y02A 50/30 20180101; A61P 31/12 20180101; A61P 31/20 20180101; C12N
9/22 20130101; A61K 38/00 20130101; C12N 15/907 20130101; A61P
31/18 20180101; Y02A 50/467 20180101; A61P 43/00 20180101; C12N
9/16 20130101; C12N 2820/60 20130101; C12N 2310/20 20170501; A61P
31/22 20180101; Y02A 50/387 20180101; Y02A 50/463 20180101; C12Y
301/00 20130101; C12N 15/86 20130101; C12N 2810/60 20130101; Y02A
50/385 20180101; A61K 48/005 20130101; C12N 2330/51 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/113 20060101 C12N015/113 |
Claims
1. A composition, wherein said composition is a CRISPR/Cas9/gRNA
complex comprising guide RNAs for use in the treatment of latent
Herpes viruses within a cell.
2-3. (canceled)
4. The composition of claim 1, wherein the latent Herpes viruses
are Epstein-Barr viruses (EBV).
5. The composition of claim 1, wherein the composition causes
insertions, deletions, or rearrangements within the viral genome in
order to incapacitate or destroy the virus.
6. The composition of claim 4, wherein the CRISPR/Cas9/gRNA complex
targets an Epstein-Barr virus (EBV) genomic region selected from
the group consisting of EBNA1, EBNA3C, Pst1 repeats, EBNA-LP
repeats and 125 bp repeats.
7. A composition comprising a CRISPR/Cas9 plasmid comprising or
consisting of a U6 promoter driven chimeric guide RNA (sgRNA) and a
ubiquitous promoter driven Cas9.
8. The composition of claim 7, wherein the composition is
transfected into cells by a viral vector or a non-viral vector.
9. The composition of claim 7, wherein the plasmid comprises an EBV
origin of replication (EBV oriP).
10. A composition comprising guide RNAs that target an Epstein-Barr
virus (EBV) genomic region selected from the group consisting of
EBNA1, EBNA3C, LMP1, PstI repeats, EBNA-LP repeats and 125 bp
repeats.
11. The composition of claim 10, wherein the guide RNAs target the
following Epstein-Barr virus (EBV) genomic repeat regions: PstI
repeats, EBNA-LP repeats and 125 bp repeats.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] The present application is a Continuation of
PCT/US2015/033199 filed May 29, 2015; which claims priority to, and
the benefit of, U.S. Provisional Patent Appln. Nos. 62/029,072
filed Jul. 25, 2014, and 62/005,395 filed May 30, 2014; the
contents of which are incorporated by reference in their entirety
for all purposes.
FIELD OF THE INVENTION
[0002] The invention generally relates to delivery methods,
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 of the infected individual. 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 virus. 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.
[0007] A newer innovative treatment is the use of nucleases to make
sequence specific deletions in the viral genome. Although this
treatment shows promise, one of the major challenges of this and
other targeted therapies is how to effectively deliver the
treatment to the cells of interest.
[0008] Some viral infections affect only a small number of cells,
and so a general non-targeted delivery approach would be
ineffective. It has been estimated that HSV, for example, latently
infects only 20,000 neurons. For this and other viral infections,
it is important to have a treatment that is targeted only to the
cells of interest. Increasing the cell affinity and specificity can
greatly improve therapeutic delivery efficiency.
SUMMARY
[0009] The invention generally relates to compositions and methods
for delivery of antiviral therapeutics. Methods and compositions
are provided for targeted delivery of antiviral therapeutics into
cells of interest using, for example, viral vectors such as
adenovirus, AAV, and replication incompetent HSV. These and other
delivery systems can be used as vehicles to deliver nucleic acid
(DNA, RNA, synthetic nucleic acids, such as PNA, LNA, etc) vectors
encoding a nuclease or a cytotoxic genetic cassette. Delivery
methods of the invention are useful to deliver vectors containing
antiviral gene editing sequences. The invention also contemplates
delivering naked DNA or RNA, protein products, plasmids containing
a promoter or other regulatory sequence that is active only in a
latent viral state which controls a cell-killing genetic construct,
or expression of a therapeutic agent (e.g., a cytotoxic
protein).
[0010] The invention also generally relates to compositions and
methods for selectively treating viral infections using a guided
nuclease system. In general terms, compositions of the invention
comprise a guide RNA that targets viral genomic material for
destruction by the nuclease and does not target the host cell
genome.
[0011] Certain embodiments of the invention make make use of a
CRISPR/Cas9 nuclease and guide RNA (gRNA) that together target and
selectively edit or destroy viral genomic material. 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. In a preferred
embodiment, the guide RNA is 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.
[0012] The presented methods provide for a CRISPR/gRNA/Cas9 complex
or other therapeutic agents to be delivered to a cell (including
entire tissues) that is infected by a virus. The CRISPR/gRNA/Cas9
complexes of the invention can be delivered by viral, non-viral or
other vectors. Viral vectors include retrovirus, lentivirus,
adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia virus or
adeno-associated viruses. Delivery can also be accomplished by
non-viral vectors, such as nanoparticles, cationic lipids, cationic
polymers, metallic nanoparticles, nanorods, liposomes, micelles,
microbubbles, cell-penetrating peptides, or lipospheres. Some
non-viral vectors may be coated with polyethyleneglycol (PEG) to
reduce the opsonization and aggregation of non-viral vectors and
minimize the clearance by the reticuloendothelial system, leading
to a prolonged circulation lifetime after intravenous
administration. Aspects of the invention provide for the
application of energy to delivery vectors for increased
tissue-permeabilizing effects (e.g., ultrasound). The invention
contemplates both systemic and localized delivery.
[0013] Aspects of the invention allow for CRISPR/gRNA/Cas9
complexes to be designed to target viral genomic material and not
genomic material of the host. 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.
[0014] 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. Aspects of the
invention involve providing a CRISPR/gRNA/Cas9 complex that
selectively targets 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.
The presented methods allows for treatment targeted disruption of
viral genomic function or, in a preferred embodiment, digestion of
viral nucleic acid via multiple breaks caused by targeting multiple
sites for endonuclease action in the viral genome. Aspects of the
invention provide for transfection of a CRISPR/gRNA/Cas9 complex
cocktail to completely suppress cell proliferation and/or induce
apoptosis in infected cells. Additional aspects and advantages of
the invention will be apparent upon consideration of the following
detailed description thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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 C.sub.t values from single cells before treatment.
(FIG. 4E) Histogram of EBV quantitative PCR C.sub.t values from
single live cells 7 days after sgEBV1-7 treatment. Red dash lines
in (FIG. 4D) and (FIG. 4E) represent C.sub.t values of one EBV
genome per cell.
[0019] 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.
[0020] FIG. 6 represents CRISPR cytotoxicity test with EBV-negative
Burkitt's lymphoma DG-75.
[0021] FIG. 7 represents CRISPR cytotoxicity test with primary
human lung fibroblast IMR-90.
[0022] FIG. 8 shows a table (Table 51) of guide RNA target
sequences for EBV.
[0023] FIG. 9 depicts Table S2.
[0024] FIG. 10 shows a CRISPR guide RNA with various targets along
the EBV reference genome. Green, red and blue represent three
different target sequence categories.
[0025] FIG. 11 shows the results of EBV-positive Raji cells
transduced with GFP-Cas9 and three different EBV-targeting guide
RNA sequences with the mCherry reporter gene, all packaged with
three adenovirus lines. Five days after transduction, GFP-mCherry
double positive cells (P9) revealed apoptotic signals with positive
Annexin V Alexa647 staining, whereas single positive cells (P7 and
P8) showed no Annexin V staining difference from uninfected double
negative cells (P6). This demonstrates that viral mediated CRISPR
delivery results in reactivation of host apoptosis pathways.
[0026] FIG. 12 shows the results of a similar CRISPR transduction
experiment. Nine days after CRISPR adenovirus transduction, PCR
assays revealed sequence specific large deletions in P9, but not P7
cells. Lane 1 and 2 are 3F/5R PCR amplicons from P7 and P9. Lane 4
and 5 are 4F/5R PCR amplicons from P7 and P9. Genome context around
guide RNA sgEBV3/4/5 and PCR primer locations are shown above.
Therefore viral mediated CRISPR delivery successfully ablates the
EBV genome.
DETAILED DESCRIPTION
[0027] The invention generally relates to compositions and methods
for delivery of therapies targeting viral infection including
compositions and methods for selectively targeting viral infections
using a guided nuclease system. The invention provides methods and
compositions that to allow effective delivery of nucleases or other
cytotoxic elements to cells of interest. Methods and compositions
are provided for targeted delivery of antiviral therapeutics into
cells of interest using, for example, viral vectors such as
adenovirus, AAV, and replication incompetent HSV. These and other
delivery systems can be used as vehicles to deliver DNA vectors
encoding a nuclease or a cell-killing gene. These delivery methods
can also be used to deliver naked DNA or RNA, protein products,
plasmids containing a promoter that is active only in a latent
viral state which drives a cell-killing gene, or other therapeutic
agents. Methods and compositions of the invention are designed to
specifically target virus and virus-infected cells.
[0028] One of the treatments contemplated by the invention is the
use of nucleases to target viral genomes. In some embodiments, the
invention involves delivering a nuclease into a cell of interest.
Nucleases have the ability to incapacitate or disrupt latent
viruses within a cell by systematically causing deletions in the
viral genome, thereby reducing the ability for the viral genome to
replicate itself. In embodiments, the treatment comprises
CRISPR/Cas and guided RNA complexes, which cause insertions,
deletions, or rearrangements within the viral genome in order to
incapacitate or destroy the virus.
[0029] Methods of the invention can be used to incapacitate or
disrupt latent viruses within a cell by systematically causing
large or repeated deletions in the genome, reducing the probability
of reconstructing the full genome. In alternative embodiments, the
CRISPR/Cas and guided RNA complexes cause insertions, deletions, or
rearrangements within the viral genome in order to incapacitate or
destroy the virus. 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 by
introducing 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). Additionally, in the co-pending U.S. Provisional
Application 62/005,395 it has been proposed as an anti-viral
therapy or more broadly as a way to disrupt genomic material.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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 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 specific nucleotide changes at
the Cas9 induced double strand break.
[0037] 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.
[0038] 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.
[0039] As versatile as the Cas9 protein is (as either a nuclease,
nickase or platform), it requires the targeting specificity of a
gRNA in order to act. As discussed below, guide RNAs or single
guide RNAs are specifically designed to target a virus genome.
[0040] A CRISPR/Cas9 gene editing complex of the invention works
optimally with a guide RNA that targets the viral genome. Guide RNA
(gRNA) or single guide RNA (sgRNA) leads the CRISPR/Cas9 complex to
the viral genome in order to cause viral genomic disruption.
[0041] 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.
[0042] 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.
[0043] 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. A marker such as EGFP fused after the Cas9 protein
allows selection of Cas9-positive cells (FIG. 1A).
[0044] 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.
[0045] 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 (FIGS. 1C and 10 and Table S1 at FIG. 8). 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.
[0046] 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.
[0047] Prepackaged GFP-Cas9-adenovirus is available from Vector
Biolabs (Philadelphia, Pa.). Various targeting gRNA sequences, such
as sequences that target EBV can be packaged to adenovirus lines.
The gRNA sequences can be housed together with the CRISPR/Cas9
complex or separately.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] Nucleases are not the only possible therapeutic agents one
could deploy against latent viral infections. In cases where a
small number of cells are infected and it would suffice to ablate
the entire cell (as well as the latent viral genome), an aspect of
the invention contemplates administration of a vector containing a
promoter which is active in the latent viral state, wherein the
promoter drives a cell-killing gene. HSV is a particularly
interesting target for this approach as it has been estimated that
only 20,000 neurons are latently infected. (Schiffer et al).
Examples of cell-killing genes include apoptosis effectors such as
BAX and BAK and proteins that destroy the integrity of the cell or
mitochondrial membrane, such as BCL-2 and alpha hemolysin. (Bayles,
"Bacterial programmed cell death: making sense of a paradox,"
Nature Reviews Microbiology 12 pp. 63-69 (2014)). Having a promoter
that is only activated in latently infected cells could be used not
only in this context but also be used to increase selectivity of
nuclease therapy by making activity specific to infected cells; an
example of such a promoter is LAP1. (Preston and Efstathiou,
"Molecular Basis of HSV Latency and Reactivation", in Human
Herpesviruses: Biology, Therapy and Immunoprophylaxis 2007.)
[0054] These agents can be delivered either as part of a viral
vector (examples further described below), or as naked as DNA or
RNA. Naked nucleic acids can be modified to avoid degradation.
[0055] Another possibility is to deliver the protein product itself
either fused to a signaling molecule or packaged into a vesicle
with signaling molecules on surface, or packed into a nanoparticle,
vesicle, or attached to a colloid. Examples of this method of
delivery have been previously explored in cancer but not applied to
local delivery against latent viral infections. (See Alexis et al,
"Nanoparticle Technologies for Cancer Therapy" in Drug Delivery,
Handbook of Experimental Pharmacology 197, 2010.) Other delivery
methods are described in detail below. For HSV and other viruses
which are highly localized in terms of which cells and tissues they
infect, these therapies might be delivered as a local injection or
as a cream.
[0056] In other embodiments of the invention, physical approaches
can be used to ablate cells that have latent infection, taking
advantage of the fact that these are localized in diseases such as
HSV. One approach is to image infected cells (for example, using
fluorescent markers against viral protein, or against viral genome,
or fluorescence proteins induced by viral latency promoters) and
then use heat, light, or radio frequency radiation to ablate those
cells. Direct contrast agents can also be used towards infected
cells. Instead of fluorescence molecules, semiconductor or metallic
nanoparticles, colloids, or other structures that interact strongly
with light or radio frequency can be used. These can be applied
locally with a cream or injections. These substances can
potentially take advantage of a cooperative effect, such that
infected cells attract multiple particles, thereby having the
highest effect. Similar approaches have been used in cancer
treatment (See for example Jain et al "Gold nanoparticles as novel
agents for cancer therapy," Br J Radiol February 2012
85(1010):101-113). The present invention applies these techniques
to treatment of latent viral infection.
[0057] In another embodiment, labeled and infected cells can be
excised using microsurgery tools such as a fiber optic endoscope,
which allows imaging and delivery of radiation in a highly
localized manner, with single cell resolution. (See Barretto R P
and Schnitzer M J. "In Vivo Optical Microendoscopy for Imaging
Cells Lying Deep within Live Tissue." Cold Spring Harb Protoc.
2012(10) and Llewellyn M E, Barretto R P J, Delp S L &
Schnitzer M J. (2008) Minimally invasive high-speed imaging of
sarcomere contractile dynamics in mice and humans. Nature. 454
784-788).
[0058] Aspects of the invention involve introducing or delivering a
therapeutic agent, such as the CRISPR/Cas9/gRNA complex, or any of
the therapeutic agents described herein, into a cell of interest.
It should be appreciated that agents can be introduced into cells
in an in vitro model or an in vivo model.
[0059] 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 (AAV). 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.
[0060] 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
WIPO Patent Application WO/2007/071994, the contents of which are
incorporated by reference.
[0061] 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.
[0062] 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.
[0063] As opposed to lentiviruses, adenoviral DNA does not
integrate into the genome and is not replicated during cell
division. Adenovirus and the related AAV can be used 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.
[0064] 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, micelles,
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 of Felgner, WO
91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or
ex vivo administration) or target tissues (e.g. in vivo
administration).
[0065] 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.
[0066] 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 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.
[0067] 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.
[0068] 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.
[0069] In some embodiments of the invention, liposomes are used to
effectuate transfection into a cell or tissue. A "liposome" as used
herein refers to a small, spherical vesicle composed of lipids,
particularly vesicle-forming lipids capable of spontaneously
arranging into lipid bilayer structures in water with its
hydrophobic moiety in contact with the interior, hydrophobic region
of the bilayer membrane, and its head group moiety oriented toward
the exterior, polar surface of the membrane. Vesicle-forming lipids
have typically two hydrocarbon chains, particularly acyl chains,
and a head group, either polar or nonpolar. Vesicle-forming lipids
are either composed of naturally-occurring lipids or of synthetic
origin, including the phospholipids, such as phosphatidylcholine,
phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol,
and sphingomyelin, where the two hydrocarbon chains are typically
between about 14-22 carbon atoms in length, and have varying
degrees of unsaturation. The above-described lipids and
phospholipids whose acyl chains have varying degrees of saturation
can be obtained commercially or prepared according to published
methods. Other suitable lipids for use in the composition of the
present invention include glycolipids and sterols such as
cholesterol and its various analogs which can also be used in the
liposomes.
[0070] Similar to a liposome, a micelle is a small spherical
vesical composed of lipids, but is arranged as a lipid monolayer,
with the hydrophilic head regions of the lipid molecules in contact
with surrounding solvent, sequestering the hydrophobic single-tail
regions in the center of the micelle. This phase is caused by the
packing behavior of single-tail lipids in a bilayer.
[0071] 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.
[0072] 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).
[0073] 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.
[0074] 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.,
Cardiovasc Ultrasound. 2004; 2: 23, doi: 10.1186/1476-7120-2-23. In
ultrasound-triggered drug delivery, tissue-permeabilizing effect
can be potentiated using ultrasound contrast agents, gas-filled
microbubbles. The use of microbubbles as nucleic acidsvectors 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.
[0075] 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.
[0076] 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.
[0077] 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. Davis M
E, Chen Z G, Shin D M. Nat Rev Drug Discov. 2008; 7:771-782. Long
circulating macromolecular carriers such as liposomes, can exploit
the enhanced permeability and retention effect for preferential
extravasation from tumor vessels. See Biomaterials. 1995 January;
16(2):145-8. 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.
[0078] 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 et
al. issued Nov. 14, 1995 which discloses parenterally administrable
liposome formulation comprising synthetic lipids; U.S. Pat. No.
5,580,571, issued Dec. 3, 1996 to Hostetler et al. which discloses
nucleoside analogues conjugated to phospholipids; U.S. Pat. No.
5,626,869 to Nyqvist et al. issued May 6, 1997 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.
[0079] Liposomes and polymerosomes can contain a plurality of
solutions and compounds.
[0080] 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 Languir 2005, 21,
9183-9186, Lorenceau et al. "Generation of Polymerosomes from
Double-Emulsions."
[0081] 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.
[0082] 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).
[0083] 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 preferred embodiments, the CRISPR/Cas9/gRNA complexes
target latent viral genomes, thereby reducing the chances of
proliferation. The guided RNA complexes target a determined number
of categories of sequences of the viral genome 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.
[0084] After introduction into a cell, the CRISPR/Cas9/gRNA
complexes act on the viral genome. 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.
[0085] In a preferred embodiment of the invention, CRISPR/Cas9/gRNA
complexes are transfected into cells containing viral genomes. The
gRNAs are designed to localize the Cas9 endonuclease at several
locations along the viral genome. The Cas9 endonuclease caused
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. Engineered viral
particles with higher cell affinity (e.g. RGD knob) and specificity
could greatly improve delivery efficiency. Delivery of circular
instead of linear DNA may also be beneficial since the circular DNA
can replicate as episomes with replication origins.
[0086] 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, micelles,
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.
[0087] Also the following items are within the ambit of the
invention:
[0088] The first item relates to a therapeutic composition for
treatment of a viral infection, the composition comprising:
[0089] a CRISPR/Cas9 endonuclease; and
[0090] a guide RNA that specifically targets a portion of a viral
genome.
[0091] Item two is a preferred embodiment of the first item,
wherein said CRISPR/Cas9 endonuclease and said guide RNA are
co-expressed in a host cell infected by a virus.
[0092] Item three is a preferred embodiment of the first item and
of item two, wherein said CRISPR/Cas9 endonuclease and said guide
RNA are packaged in a delivery vector.
[0093] Item four is a preferred embodiment of item three, wherein
the delivery vector is a viral vector.
[0094] Item five is a preferred embodiment of item four, wherein
the viral vector is selected from the group consisting of
retrovirus, lentivirus, adenovirus, herpesvirus, poxvirus,
alphavirus, vaccinia virus and adeno-associated viruses.
[0095] Item six is a preferred embodiment of item three, wherein
the delivery vector is a non-viral vector.
[0096] Item seven is a preferred embodiment of item six, wherein
the non-viral vector is selected from the group consisting of a
nanoparticle, a cationic lipid, a cationic polymer, a metallic
nanoparticle, a nanorod, a liposome, microbubbles, cell-penetrating
peptide, and a liposphere.
[0097] Item eight is a preferred embodiment of item seven, wherein
the non-viral vector comprises polyethyleneglycol (PEG).
[0098] A ninth item relates to a method for treating a viral
infection, the method comprising the steps of:
[0099] delivering to a virus-infected cell a complex comprising a
CRISPR/Cas9 endonuclease and a guide RNA that specifically targets
one or more portions of the genome of said virus; wherein said
complex binds to and alters said viral genome but does not alter
genomic material of said infected cell.
[0100] Item ten is a preferred embodiment of item nine, wherein
said virus is latent in said virus-infected cell.
[0101] Item eleven is a preferred embodiment of item ten, wherein
the delivering step comprises delivering said complex in viral
vector.
[0102] Item twelve is a preferred embodiment of item eleven,
wherein the viral vector is selected from the group consisting of
retrovirus, lentivirus, adenovirus, herpesvirus, poxvirus,
alphavirus, vaccinia virus and adeno-associated viruses.
[0103] Item thirteen is a preferred embodiment of item ten, wherein
the delivering step comprises delivering said complex in a
non-viral vector.
[0104] Item fourteen is a preferred embodiment of item thirteen,
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.
[0105] Item fifteen is a preferred embodiment of item thirteen,
wherein the non-viral vector comprises polyethyleneglycol
(PEG).
[0106] Item sixteen is a preferred embodiment of item eleven or
thirteen, wherein the method further comprises the step of applying
energy to said vector.
[0107] Item seventeen is a preferred embodiment of item sixteen,
wherein the energy is ultrasound or electrophoresis.
[0108] Item eighteen is a preferred embodiment of item nine through
seventeen, wherein alters comprises causing a double strand break
in the genome of said virus.
[0109] Item nineteen is a preferred embodiment of item nine through
seventeen, wherein alters comprises causing multiple double strand
breaks in the genome of said virus.
[0110] Item twenty is a preferred embodiment of item nine through
nineteen, wherein alters comprises causing an insertion in the
genome of said virus.
[0111] Item twenty-one is a preferred embodiment of item nine
through nineteen, wherein alters comprises causing multiple
insertions in the genome of said virus.
[0112] Also the following aspects and embodiments are within the
ambit of the invention:
[0113] A first aspect relates to a composition for treating a
latent viral infection, the composition comprising:
[0114] a vector;
[0115] a CRISPR/Cas9 endonuclease; and
[0116] a guide DNA operable to target a portion of a viral
genome.
[0117] Embodiment two is a preferred embodiment of the first
aspect, wherein the vector is a viral vector.
[0118] Embodiment three is a preferred embodiment of embodiment
two, wherein the viral vector is selected from a group consisting
of retrovirus, lentivirus, adenovirus, herpesvirus, poxvirus,
alphavirus, vaccinia virus, and adeno-associated viruses.
[0119] Embodiment four is a preferred embodiment of the first
aspect, wherein the vector is a non-viral vector.
[0120] Embodiment five is a preferred embodiment of the fourth
embodiment, wherein the non-viral vector is selected from a group
consisting of nanoparticles, cationic lipids, cationic polymers,
metallic nanoparticles, nanorods, liposomes, micelles,
microbubbles, cell-penetrating peptides, and lipospheres.
[0121] Embodiment six is a preferred embodiment of the first
aspect, including embodiments two to five, wherein the viral genome
is an EBV genome.
[0122] Embodiment seven is a preferred embodiment of the first
aspect, including embodiments two to six, wherein the CRISPR/Cas9
endonuclease comprises circular DNA.
[0123] Embodiment eight is a preferred embodiment of the first
aspect, including embodiments two to six, wherein the CRISPR/Cas9
endonuclease comprises linear DNA.
[0124] Embodiment nine is a preferred embodiment of the first
aspect, including embodiments two to eight, wherein the composition
further comprises a promoter operable to be activated only in a
latent viral state cell.
[0125] A second aspect relates to a method for delivering a
therapeutic agent to a cell, the method comprising:
[0126] providing a composition comprising a vector, a CRISPR/Cas9
endonuclease, and a guide DNA that targets a portion of the viral
genome; and
[0127] delivering, using a vector, the composition into a cell
containing latent viral nucleic acid.
[0128] Embodiment eleven is a preferred embodiment of the second
aspect, wherein the composition is delivered using a viral
vector.
[0129] Embodiment twelve is a preferred embodiment of embodiment
eleven, wherein the viral vector is selected from a group
consisting of retrovirus, lentivirus, adenovirus, herpesvirus,
poxvirus, alphavirus, vaccinia virus, and adeno-associated
viruses.
[0130] Embodiment thirteen is a preferred embodiment of the second
aspect, including embodiments eleven to twelve, wherein the
composition is delivered using a non-viral vector.
[0131] Embodiment fourteen is a preferred embodiment of embodiment
thirteen, wherein the non-viral vector is selected from a group
consisting of nanoparticles, cationic lipids, cationic polymers,
metallic nanoparticles, nanorods, liposomes, micelles,
microbubbles, cell-penetrating peptides, and lipospheres.
[0132] Embodiment fifteen is a preferred embodiment of the second
aspect, including embodiments eleven to fourteen, wherein the viral
genome is an EBV genome.
[0133] Embodiment sixteen is a preferred embodiment of the second
aspect, including embodiments eleven to fifteen, wherein the
CRISPR/Cas9 endonuclease is circular DNA.
[0134] Embodiment seventeen is a preferred embodiment of the second
aspect, including embodiments eleven to fifteen, wherein the
CRISPR/Cas9 endonuclease is linear DNA.
[0135] Embodiment eighteen is a preferred embodiment of the second
aspect, including embodiments eleven to seventeen, wherein the
method further comprises a promoter operable to be activated only
in a latent viral state cell.
[0136] A third aspect relates to a composition for treating a
latent viral infection, the composition comprising:
[0137] a vector;
[0138] a nucleic acid comprising a promoter operable to be
activated only in a latent viral state cell; and
[0139] a cell-killing gene that is driven by the promoter.
[0140] Embodiment twenty is a preferred embodiment of the third
aspect, wherein the cell-killing gene is selected from a group
consisting of BAX, BAK, BCL-2, and alpha-hemolysin.
[0141] Embodiment twenty-one is a preferred embodiment of the third
aspect, wherein the composition is delivered using a viral
vector.
[0142] Embodiment twenty-two is a preferred embodiment of
embodiment twenty-one, wherein the viral vector is selected from a
group consisting of retrovirus, lentivirus, adenovirus,
herpesvirus, poxvirus, alphavirus, vaccinia virus, and
adeno-associated viruses.
[0143] Embodiment twenty-three is a preferred embodiment of the
third aspect, wherein the composition is delivered using a
non-viral vector.
[0144] Embodiment twenty-four is a preferred embodiment of
embodiment twenty-three, wherein the non-viral vector is selected
from a group consisting of nanoparticles, cationic lipids, cationic
polymers, metallic nanoparticles, nanorods, liposomes, micelles,
microbubbles, cell-penetrating peptides, and lipospheres.
Examples
[0145] 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.
[0146] 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).
[0147] 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 fl 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.
[0148] 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).
[0149] 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 51). EBNA1 is crucial for many EBV functions
including gene regulation and latent genome replication. We
targeted guide RNA sgEBV4 and sgEBVS 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.
[0150] 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).
[0151] DNA sequences flanking sgRNA targets were PCR amplified with
Phusion DNA polymerase (FIG. 9, Table S2). 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.
[0152] 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).
[0153] 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.
[0154] 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 (FIGS. 3E-G). Bright field
microscopy showed obvious apoptotic cell morphology (FIGS. 3H-I)
and fluorescent staining demonstrated drastic DNA fragmentation
(FIGS. 3J-M). Altogether this evidence suggests restoration of the
normal host cell apoptosis pathway after EBV genome
destruction.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] FIGS. 10 and 8 show seven CRISPR guide RNAs designed to
target the EBV genome. Pre-packaged GFP-Cas9 adenovirus
(Ad-GFP-Cas9) was purchased from Vector Biolabs. Three different
EBV-targeting guide RNA sequences (sgEBV3/4/5) were packaged into
three adenovirus lines respectively, all with mCherry reporter.
EBV-positive Raji cells transduced with GFP-Cas9 and
mCherry-sgEBV3/4/5 adenoviruses demonstrated the CRISPR-specific
apoptosis (FIG. 11) and large DNA deletions as the EBV genome was
ablated (FIG. 12) in only the GFP-mCherry double-positive
subpopulation.
INCORPORATION BY REFERENCE
[0159] 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
[0160] 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.
Sequence CWU 1
1
24120DNAArtificial 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 20820DNAArtificial
SequenceSynthetic sgEBV1F PCR Primer 8tgctaggcca ccttctcagt
20920DNAArtificial SequenceSynthetic sgEBV1R PCR Primer 9gtagtgtgtg
cctgggtgtg 201020DNAArtificial SequenceSynthetic sgEBV2F PCR Primer
10agcatggcga agtagacagg 201118DNAArtificial SequenceSynthetic
sgEBV2R PCR Primer 11gcccattcga accctacc 181220DNAArtificial
SequenceSynthetic sgEBV3F PCR Primer 12tttcagaccc accatggaat
201320DNAArtificial SequenceSynthetic sgEBV3R PCR Primer
13cccatgaacc cagttagagg 201420DNAArtificial SequenceSynthetic
sgEBV4F PCR Primer 14ggctgcgagt aattggtgat 201521DNAArtificial
SequenceSynthetic sgEBV4R PCR Primer 15caatgcaact tggacgtttt t
211620DNAArtificial SequenceSynthetic sgEBVSF PCR Primer
16gctgaggttt tgaaggatgc 201720DNAArtificial SequenceSynthetic
sgEBVSR PCR Primer 17ggagctgagt gacgtgacaa 201820DNAArtificial
SequenceSynthetic sgEBV7F PCR Primer 18agtaagggaa agggggtgtg
201920DNAArtificial SequenceSynthtic sgEBV7R PCR Primer
19gacgtagccg ccctacataa 202020DNAArtificial SequenceSynthetic
oriP_F PCR Primer 20ccaccaattc caaccatttt 202115DNAArtificial
SequenceSynthetic oriP_R PCR Primer 21cgcggggcag tgcat
152220DNAArtificial SequenceSynthtic EBNA1_qF PCR Primer
22cctccctggt ttccacctat 202320DNAArtificial SequenceSynthetic
EBNA1_qR PCR Primer 23cctccttcat ctccgtcatc 202418DNAArtificial
SequenceSynthetic EBNA1_qP PCR Primer 24tccgtcatca ccctccgc 18
* * * * *