Gene TarGeTinG and Gene CorreCTion i
mutation and correction of SMN2 aberrant splicing, by exploiting the
Conclusions
non-homologous end-joining (NHEJ) pathway. Plasmids encoding
We have generated a series of combination RNA knockdown and
Cas9-GFP under the control of CMV promoter, and selected gRNAs replacement AAV vectors that may be useful for the treatment of
downstream to the Pol-III U6 promoter (Addgene) were transfected ADRP. At early time points, our tests of these specific vectors have
in HEK-293T cell line and in immortalized myoblasts derived from not been conclusive. The injected mice will be followed for longer
either healthy donors or SMA patients. Transfection efficiency was intervals and additional mice will be added to the study to determine
estimated as percentage of GFP-expressing cells (20-50% and 1-10%, if the difference in visual function of the experimentally treated eyes
respectively) and nuclease activity detected by Surveyor assay versus the control is statistically significant.
and target site sequencing. In particular, in SMA patient-derived
myoblasts we detected mutations (indels) at the level of the induced
1
33.
Use of 2As To Control Protein Subcellular
DNA double-strand break at ~30% frequency. Levels of SMN
restoration will be investigated by qPCR of the different species of
SMN transcripts and by western blotting of SMN protein. The goal
of this study is to provide an in vitro proof of principle of effective
gene correction in SMA patient-derived cells. In the context of a
multisystemic, complicated disease such as SMA, targeted genome
editing strategy could represent an additional therapeutic tool.
Localization
Ekaterina Minskaia, Claire Roulston, Garry Luke, Martin D.
1
1
1
1
Ryan.
1
Biomedical Sciences Research Complex (BSRC), University of St.
Andrews, St. Andrews, United Kingdom.
A substantial proportion (~39%) of all human proteins are either
secreted from the cell, located within the lumen/ membranes of
cytoplasmic vesicular structures, or, are plasma membrane proteins.
Given that such high proportion of proteins are initially translocated
into the endoplasmic reticulum (ER), many therapeutic strategies
rely on the ability to co-express multiple proteins – some, or all of
which, might be targeted to such sites. This directly applies to in vivo
gene therapy strategies, or, when therapeutic proteins may need to be
co-expressed with selectable markers (e.g. ex vivo gene therapies).
As was shown before, Picornavirus 2A (foot-and-mouth disease
virus 2A; F2A) and ‘2A-like’sequences are powerful tools that allow
multiple proteins to be translated and co-expressed from a single
transcript mRNA under the control of only one promoter. When
132.
Gene Therapy With Self-Complementary
Recombinant Adeno-Associated Virus in Models of
Autosomal Dominant Retinitis Pigmentosa Cause
by RHO Mutations
1
1
1
1
Brian Rossmiller, Danny Zakria, Arathi Nandyala, Hiral Jivanji,
1
Lewin Alfred.
1
Molecular Genetics and Microbiology, The University of Florida,
Gainesville, FL.
Purpose
Retinitis pigmentosa is the leading hereditary cause of blindness
with 30-40% of cases attributable to autosomal dominant retinitis
pigmentosa (ADRP). ADRP arises from mutations in at least 24
known genes with 30% arising in the rhodopsin gene (RHO). Given
the large heterogeneity of mutations in RHO leading to ADRP, we
propose knocking down of endogenous RHO and replacing it with
a “hardened” copy, or a RHO with nucleotide changes that preserve
the amino acid sequence but decrease the efficiency of knock-down.
Here we report the use of a scAAV serotype 8 (Y733F) to express a
hardened human rhodopsin (hRHO) under the control of the human
opsin proximal promoter (HOPS) and an H1 promoter driven shRNA.
Methods
2
A is positioned between sequences encoding two, or more, genes,
it mediates a co-translational ‘cleavage’ at its own C-terminus. A
major problem with co-expression of certain proteins targeted to, or
transiting through, the ER is that the ‘cleavage’activity of short F2As
can be greatly inhibited by sequences immediately upstream leading
to aberrant sub-cellular localisation of some proteins.
We have also discovered a number of active cellular 2A-like
sequences, associated with non-long terminal repeat (non-LTR)
retrotransposons, but also with structural and metabolic proteins:
ankyrin repeats, sodium dependent neutral amino acid transporters,
and NOD-like receptor (NLR) proteins. Examination of the
surrounding protein and gene structure revealed that in the majority
of cases these 2A sequences occurred as N-terminal features.
Interestingly, using SignalP, many of these novel 2As scored highly
as N-terminal signal peptides.
Four different knock-down methods were tested, ribozyme (Rz)
4
07 and Rz525, miRNA 301 and shRNA 301 against both the wild-
type and hardened RHO target regions. The transfections were done
in HEK293 cells (n=6) with (1) the target plasmid psiCheck2 dual
luciferase containing RHO target region, (2) plasmid expressing the
shRNA, miRNAor ribozyme against the target region and (3) a control
miRNA. The reduction in expression of luciferase was measured at
Here, we present our latest findings on 2Asequences.Aseries of test
proteins (eGFPand mCherry) were expressed i) followed by ‘hybrid’
‘self-cleaving’F2Asequences (with different upstream contexts) or ii)
downstream of a putative signal 2A. We demonstrate that inhibition
of F2A-mediated cleavage in shorter sequences can be overcome by
introduction of mutations upstream of 2A changing the context of
the sequence between the C-terminus of the upstream protein and 2A
sequence. In the case of N-terminal - cellular - (NLR) 2As, ‘uncleaved’
2
4 and 48 hours post transfection.
We are using adeno-associated virus vectors to deliver these
RNA knockdown agents to mouse models of ADRP: Rho I307N
and human RHO transgenic T17M and P23H. Mice were injected
with AAV-HOP-hRHO and knockdown agents at postnatal day
2Aindeed can act as a signal peptide. If 2Adoes not ‘cleave’, it directs
5
. I307N Rho mice were created through the use of N-ethyl-N-
a proportion of the newly synthesised reporter protein to the exocytic
pathway: if 2A ‘cleaves’, the protein downstream is localised to the
cytoplasm. This type of 2A mediates, therefore, a newly discovered
form of dual protein targeting.
nitrosourea (Budzynski et al. JBC 2010). The I307N mouse model
exhibits very slow degeneration under ambient light but is reduced
in visual response to light by 50% in one week post exposure to
1
0,000 lux. Intravitreal injections with one of the two constructs,
hRHO+shRNA301, or hRHO+shRNA750 in one eye and AAV-
HOPS-mCherry or sham injection in the other. The mice were
followed using electroretinogram and optical coherence tomography.
Results
The knock-down results show shRNA301 and ribozyme 525
to cause the largest reduction of RHO mRNA. At one month post
injection there was statistically significant difference between T17M
RHO eyes injected with AAV-hRHO-shRNA750 and the sham
injected eyes.
S54
Molecular Therapy Volume 23, Supplement 1, May 2015
Copyright © The American Society of Gene & Cell Therapy
Cao, Duan-lin
