Download Is there an alternative to MRT?

but it still needs a bit of work
[email protected]
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Reprogenetics
ART Institute of Washington
Life Global
Principle investigator of cytoplasmic transfer
series (1996-2001)
• Is there an alternative to MRT?
• Lessons from the cytoplasmic transfer
series
• Cellular stress from micromanipulation?
(safety)
• Preferred technical approach (MST, PNT,
PBT)?
• Nomenclature?
• YES – Preimplantation Genetic Diagnosis or PGD
(PGD for mitochondrial disease)
• Use of PGD varies from 3-85% (USA data)
• Usage in Europe mostly limited to early embryo stages (.)
• Blastocyst culture increases success rates
• Allows biopsy of a better sample
• Cryopreservation survival higher with blastocysts
• Evidence shows PGS is successful at blastocyst
• Less than 50 cases of PGD for Mito Disease
• About a dozen papers (using pb or blastomere biopsy)
• Shows reliability increases with cell and egg number
• Shows little variation in mutation load
• PGD for mito disease should be considered at blastocyst
stage in combination with vitrification
Sallevelt et al, 2013
• Mid-90s state of assisted reproduction
• Acceptable pregnancy rates achieved by multiple embryo
transfer
• Early knowledge about embryonic aneuploidy and age
• ‘Cytoplasmic failure’ seemed a reasonable proposition
• Selected patients with multiple failed IVF, poor embryo
development but…. NOT increased maternal age
• Three patient cycles using electro-fusion of cytoplasts
• Increased abnormal fertilization (same as MRT work)
• Changed technology to cytoplasmic transfer by injection
• Based on ICSI for male factor (>50,000 babies)
Cohen et al, 1997 and 1998
Cohen et al, 1998
• 37 cycles (33 patients)
• 17 babies born
• 12 deliveries
• 1 early XO miscarriage
• 1 late XO termination
• One male twin at 18 months borderline PDD-NOS
• Two babies confirmed with donor mitochondria in a
series of n=8
• ‘Heteroplasmy’
• First phase follow-up study 2014-2015
• Preliminary data is reassuring
Cohen et al, 1997, 1998, 1999; Brenner et al 2000, Barritt et al, 2001
• Two types of experiments
• Using combinations of two inbred species: heteroplasmic
mice are developmentally affected (minor to major)
• Using inter-strain and F1 hybrids: (no effect or minor)
• Inbred - At least 20 consecutive generations of sibling or
parent/offspring mating
• Inbred mice are homozygous at virtually all of their loci
• We are not an inbred species
Acton et al, 2007 – Liang et al, 2009 - Cheng et al, 2009
“We find that inter-strain mPNT, ST, and ooplasm transfer produce little or no long-term
adverse effects on growth rate or methylation states of the genes assayed.”
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Seen after all forms of manipulation
But can be minor (ICSI)
Cytoplasmic transfer (CT) similar to ICSI
CT pilot study using ICSI
(>50,000 ICSI babies in 1996)
Electrofusion - no prior alternative
(0 babies)
Sendai Virus Extract (SeV)
(0 babies)
Membrane relaxants
(0 babies)
• Two implantations with XO after CT
• Possibly significant
• Possibly related to cellular stress (XO
can occur during formation of pb2)
• Strongly recommend PGS after MRT to
confirm normal chromosome set
• Polar body transfer (PBT) was successfully used to model
MRT in the mouse (Wang et al, 2014)
• Highly successful in the mouse
• In some mouse strains polar bodies are sometimes viable (.)
• In the human they are very different from patient to patient
“Changing genes which
are passed on to future
generations” or
“changing genes in
gametes and very early
embryos” [meaning not
temporarily as in
altered gene
expression – but
changing the code].
Maybe – but the
altered genes already
existed in someone
else. It is the
combination of nDNA
and mtDNA that is
novel, but that
descriptive is similar to
each new fertilization.
“Gene therapy is an
experimental
technique that
uses genes to treat or
prevent disease. In the
future, this technique
may allow treatment of
a disorder by inserting
a gene into a patient's
cells instead of using
drugs or surgery.”
Yes, very close, maybe
a specialized form? “Gene therapy is an
experimental
technique that
uses someone else’s
genes to treat or
prevent disease.”
Applies to MRT. Does it
apply to CT?
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or Genetic Engineering.
“Genetic engineering is
the process of
manually adding new
DNA to an organism.
The goal is to add one
or more new traits that
are not already found
in that organism.”
This probably does not
apply.
No likely addition of
new traits.
Addition of new traits is
not the goal.
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