Therapeutics

1. - Satrupa Das
Dept. of Molecular Biology

2. A.
B.
Viral gene therapy
RNAi therapy

3.  Despite promise and recent success, gene therapy and RNAi have
limitations that preclude their utility for a large number of diseases.
 Viral gene therapy may cause mutagenesis at the insertion site and
result in dysregulated transgene expression.
 Meanwhile, the use of RNAi is limited to targets for which gene
knockdown is beneficial. Also, RNAi often cannot fully repress gene
expression and is therefore unlikely to provide a benefit for diseases in
which complete ablation of gene function is necessary for therapy.
RNAi may also have poor specificity, posing potential safety concerns
and sometimes decreasing the effectiveness of treatment.

4. Genome Editing
Technologies
Programmable nucleases enable precise genome editing by introducing DNA double-
strand breaks (DSBs) at specific genomic loci.
 DSBs subsequently recruit endogenous repair machinery for either
non-homologous end-joining (NHEJ) or homology-directed repair (HDR) to the DSB
site to mediate genome editing.
Four major classes of nucleases - meganucleases and their derivatives,
zinc finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs)
and CRISPR-associated nuclease Cas9 .
These nuclease systems can be broadly classified into two categories based on their
mode of DNA recognition: ZFNs, TALENs and meganucleases achieve specific DNA
binding via protein- DNA interactions, whereas Cas9 is targeted to specific DNA
sequences by a short RNA guide molecule that base-pairs directly with the target DNA
and by protein-DNA interactions.

6. Meganucleases are endoeoxyribinucleases that have a
large recognition site, which occurs rarely in entire
genome. Consequently, they can be used as highly
specific tools in genome engineering to either modify or
eliminate a particular gene. Eg. I-Sce-I
1. 2.
3. 4.

10. Duchenne muscular dystrophy (DMD) – severe muscle-degenerative disease caused
by loss-of-function mutations in dystrophin gene located on X chromosome.
Dystrophin gene consists of 79 exons and disruption of the protein reading frame by
small deletions, exon duplications or loss of exons leads to DMD.
Programmable nucleases like TALEN and CRISPR-Cas9 provide greater flexibility
than meganucleases or ZFNs with regards to selecting target regions of interest.
A DMD patient with a deletion of exon 44 dystrophin was detected.
Attempted knock-in strategy of the deleted exon 44 in front of exon 45.
iPSC lines from fibroblasts were obtained from this patient (cells having normal
karyotype and expressed pluripotency markers like TRA-1-60, SSEA5,OCT3/4 and
NANOG were chosen).
Constructed a donor template vector to conjugate exon 45 with exon 44 with a
hygromycin-selection casette flanked by 2 loxP sites.

11. Targeting donor was electroporated with TALENs/Cas9sgRNA expression vectors.
Hygromycin selection for isolation of several clones and screened for knockin clones
by PCR analysis.
Regardless of nuclease used 90% of analyzed clones showed targeting of donor
template at the target locus. But single copy knock-in clones confirmed by Southern
blot analysis.

12. To remove the selection cassette from the knockin clones transiently treated the
obtained clones with the Cre expression vector to excise the hygromycin-selection
cassette.
Successful excision confirmed by genomic PCR and Southern blotting.
Sequence the chimeric exon 44-45 in the knockin clones by Sanger sequencing, and
detected no extra mutations.
Expression of pluripotency markers in knockin clones.
To confirm that genetic correction by TALEN and CRISPR resulted in restoration of
the dystrophin gene products differentiate the original and corrected iPSC clones into
skeletal muscle cells. Check by PCR and sequencing, immunofluorescence staining
with an anti-dystrophin antibody and western-blot analysis.

13. Factors influencing therapeutic efficacy

14. Efficiency of NHEJ- and HDR-mediated DSB repair varies substantially by cell type
and cell state; in most cases, however, NHEJ is more active than HDR.
This difference in activity makes it more challenging to treat diseases that require
gene correction or gene insertion than those requiring gene inactivation .
NHEJ is thought to be active throughout the cell cycle and has been observed in a
variety of cell types, including dividing and post-mitotic cells. Therefore be used to
facilitate high levels of gene disruption in target cell populations .
Efficiency of genome editing

15. In contrast, HDR acts primarily during the S/G2 phase and is therefore largely
restricted to cells that are actively dividing, limiting treatments that require precise
genome modifications to mitotic cells .
 efficiency of correction by HDR may be controlled by a number of factors .
 First, large HDR-mediated insertions have been found to occur at a lower rate than
HDR-mediated small deletions, insertions or substitutions .
Second, the exact sequence changes made through HDR may influence therapeutic
efficacy, as editing events that do not destroy the nuclease recognition site may be
subject to further mutagenesis by NHEJ, potentially reducing therapeutic editing rates .
Third, increasing the extent of similarity between the repair template and the DSB
site may increase HDR rates.
Lastly, suppressing competing DNA repair pathways i.e NHEJ has also been shown
to increase HDR rates moderately.

16. Modes of delivery and Examples of successful genome
editing therapeutic strategies
Diseases affecting blood system such as SCID, Fanconi anemia, Wiskott-Aldrich
syndrome
HIV
1. Ex-vivo editing therapy

17. 2. In-vivo editing therapy
Haemophilia B (restoring factor IX)
Hereditary tyrosinemia

18. Challenges to clinical translataion
Safety and efficacy
Increasing efficiency of gene correction
Understanding and improving specificity of editing nucleases
Delivery