16
Mar
09

A translation

From “journalism” to science you can understand.

In case you haven’t picked up on the “breaking news” of a “DNA ‘Patch’ for Canine Form of Muscular Dystrophy,” allow me to be the one to burst their bubble.

1) The “technology” they call “exon skipping” isn’t really that new, this clinical usage is new, but it’s been used before, mainly to produce gene  knockdowns. [edit: knockout mutants require DNA to be modified]

2) They aren’t called “molecular ‘patches,'” they are called morpholinos (which they do call them much later), which are nitrogenous bases bound to a morpholine ring which prevents RNase H from degrading it. Other types of oligos have been used to produce knockdowns and knockout mutants for decades, but done via electroporation rather than transmembranous peptides.

3) In order to get these morpholinos to have any effect, they must be bound to a peptide capable of crossing the cell membrane; (or as Jon pointed out, high doses can also work) once in the cytoplasm, this peptide must release the morpholino and it will diffuse into the nucleus.

4) Because the mutations associated with muscular dystrophy are still being explored, not all types can be tested for at this time; we need to know what segment of the RNA sequence needs to be modified before being able to modify them.

In short, the “new technology” is not the morpholino itself, but the method of getting bloodstream morpholinos into the cell nucleus. We’ve been able to “patch” DNA for decades, doing so without electroporation or micro-injection with low doses is the key.

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4 Responses to “A translation”


  1. March 23, 2009 at 4:57 pm

    You’re on track. The Morpholinos used in the canine DMD study were produced by the company I work for, so I’ve a few comments.

    1) The “technology” they call “exon skipping” isn’t really that new, it’s clinical usage is new, but it’s been used before, mainly to produce knockdown/knockout mutants.

    Notes:
    Exon skipping started in Ryszard Kole’s lab.

    http://www.pnas.org/content/90/18/8673.long

    It is a form of knockdown (altered RNA), not a knockout nor a mutant (since DNA is not altered). The first clinical trial with Morpholinos was less than 10 years ago, but several trials have been conducted (e.g. cardiac restenosis, hepatitis C, West Nile virus).

    2) They aren’t called “molecular ‘patches,’” they are called morpholinos (which they do call them much later), which are oligonucleotides bound to a morpholine ring which prevents RNase H from degrading it. These are a modified oligo sequence. Oligos have been used to produce knockout/knockdown mutants for decades, but done via electroporation rather than transmembranous peptides.

    Notes:
    Morpholinos are oligomers but not oligonucleotides. Oligonucleotides by definition contain ribose or deoxyribose. Instead, as you pointed out, Morpholinos contain a morpholine ring. RNase-H degrades RNA bound to RNase-H competent oligos. Morpholinos do not trigger RNase-H activity to degrade the mRNA to which a Morpholino oligo is bound. Most use of Morpholinos has involved microinjection into embryos, though there has also been some electroporation and some reagent-mediated delivery.

    3) In order to get these morpholinos to have any effect, they must be bound to a peptide capable of crossing the cell membrane; once in the cytoplasm, this peptide must release the morpholino and it will diffuse into the nucleus.

    Notes:
    The dog study by Yokota et al. used unmodified Morpholinos (no delivery peptide). When Morpholinos are microinjected or electroporated, they are also used without peptides. However, for systemic efficacy at low doses the peptides are needed; the dog study used very high-dose injections instead. The arginine-rich cell-penetrating peptides are likely to be key to the development of future Morpholino-based therapeutics.

    4) Because the mutations associated with muscular dystrophy are still being explored, not all types can be tested for at this time; we need to know what DNA sequences need to be modified before being able to modify them.

    Notes:
    Morpholinos interact with RNA when they are used to modify splicing.

    In short, the “new technology” is not the morpholino mutation itself, but the method of getting bloodstream morpholinos into the cell nucleus. We’ve been able to “patch” DNA for decades, getting it into the cellular nucleus without electroporation or micro-injection is the key.

    Notes:
    The Morpholinos do no trigger changes in DNA, so there is no mutation associated with their use. While I agree that getting Morpholinos into the nucleus is key to their efficacy, Yokota et al. did the job by administering high doses into dystrophic dogs; dystrophy causes leakiness of muscles, allowing some Morpholino to enter.

  2. 2 jaredcormier
    March 23, 2009 at 7:02 pm

    Thank you for your corrections.
    A few comments in response:
    1) I was always under the impression that the distinction between a knockdown and a knockout was that in a knockdown, there is still SOME functional product while in a knockout there is no function. In any case, I agree with you that morpholinos are a form of knockdown, and are oligomers (rather than oligonucleotides). [I apologize for these mistakes, I wrote this rather rapidly while quite tired...]

    2) I was trying to state that “morpholinos are similar to oligonucleotides with morpholine rather than sugar” but I royally screwed that one up, thank you.

    3) Do you think it would be possible to use morpholinos for purposes of selectively knocking down production in a very specific cell type?

    4) Once again, thank you, I will be making corrections to the errors you have drawn my attention to.

  3. March 24, 2009 at 11:03 am

    Hi Jared,

    Knockdowns can be applied to DNA-level changes where transcription is reduced but not eliminated, while knockout is used to describe a DNA change resulting in elimination of transcription. Oligo-based techniques that reduce or apparently eliminate translation are all termed knockdowns.

    The problem of selectively knocking down translation of a particular mRNA in a particular cell type can be approached two ways. First, if the transcript of interest is made in the particular cell type and not in other cells, you just deliver a specific oligo everywhere and it is only active where the mRNA with the oligo’s complementary target is being transcribed. That is easy to do with current technology, but it is a special case. I think you were addressing the more difficult challenge of selectively delivering oligos only to a particular type of cell. There is not a great technology for that currently. If you only need delivery to one or a few cells, you can microinject large cells or do single-cell electroporation. For targeting a particular cell type throughout the body, there is the possibility of combining a technique like conjugation of a cell-penetrating peptide to a Morpholino with an additional conjugation of a cell-specific ligand. There is research ongoing to develop these bifunctional delivery moieties for Morpholinos, but no clear successes have been reported yet.

    The press releases that I read were not clear and had some misinformation. I’m glad you are making an effort to translate these, that’s helpful work.

    Best regards,

    – Jon


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