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is it possible to knockdown a gene in a living adult now

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is it possible to knockdown a gene in a living adult now

Postby steelcat » Thu Nov 06, 2008 9:49 am

is it possible to knockdown a gene in a living adult now?
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Postby MrMistery » Fri Nov 07, 2008 1:34 am

no.
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Re: is it possible to knockdown a gene in a living adult now

Postby jonmoulton » Fri Nov 07, 2008 3:44 pm

Yes.

Following are references related to conjugation of Morpholino oligos with arginine-rich peptides.

Zhang B, Dong H, Stein DA, Shi PY. Co-selection of West Nile virus nucleotides that confer resistance to an antisense oligomer while maintaining long-distance RNA/RNA base pairings. Virology. 2008 Oct 6. [Epub ahead of print]

Abes R, Moulton HM, Clair P, Yang ST, Abes S, Melikov K, Prevot P, Youngblood DS, Iversen PL, Chernomordik LV, Lebleu B. Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies. Nucleic Acids Res. 2008 Sep 16. [Epub ahead of print]

Bok K, Cavanaugh VJ, Matson DO, González-Molleda L, Chang KO, Zintz C, Smith AW, Iversen P, Green KY, Campbell AE. Inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome. Virology. 2008 Sep 8. [Epub ahead of print]

Yin H, Moulton HM, Seow Y, Boyd C, Boutilier J, Iverson P, Wood MJ. Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum Mol Genet. 2008 Sep 10. [Epub ahead of print]

Wu B, Moulton HM, Iversen PL, Jiang J, Li J, Li J, Spurney CF, Sali A, Guerron AD, Nagaraju K, Doran T, Lu P, Xiao X, Lu QL. Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci U S A. 2008 Sep 19. [Epub ahead of print]

Jearawiriyapaisarn N, Moulton HM, Buckley B, Roberts J, Sazani P, Fucharoen S, Iversen PL, Kole R. Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice. Mol Ther. 2008 Sep;16(9):1624-9. Epub 2008 Jun 10.

Paessler S, Rijnbrand R, Stein DA, Ni H, Yun NE, Dziuba N, Borisevich V, Seregin A, Ma Y, Blouch R, Iversen PL, Zacks MA. Inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers. Virology. 2008 Jul 5;376(2):357-70. Epub 2008 May 12.

Abes R, Arzumanov A, Moulton H, Abes S, Ivanova G, Gait MJ, Iversen P, Lebleu B. Arginine-rich cell penetrating peptides: Design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides. J Pept Sci. 2008 Apr;14(4):455-60.

Lupfer C, Stein DA, Mourich DV, Tepper SE, Iversen PL, Pastey M. Inhibition of influenza A H3N8 virus infections in mice by morpholino oligomers. Arch Virol. 2008 May;153(5):929-37. Epub 2008 Mar 28.

Patel D, Opriessnig T, Stein DA, Halbur PG, Meng XJ, Iversen PL, Zhang YJ. Peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication. Antiviral Res. 2007 Oct 4; [Epub ahead of print]

Lebleu B, Moulton HM, Abes R, Ivanova GD, Abes S, Stein DA, Iversen PL, Arzumanov AA, Gait MJ. Cell penetrating peptide conjugates of steric block oligonucleotides. Adv Drug Deliv Rev. 2008 Mar 1;60(4-5):517-29. Epub 2007 Oct 22.

Lai SH, Stein DA, Guerrero-Plata A, Liao SL, Ivanciuc T, Hong C, Iversen PL, Casola A, Garofalo RP. Inhibition of Respiratory Syncytial Virus Infections With Morpholino Oligomers in Cell Cultures and in Mice. Mol Ther. 2008 Apr 29; [Epub ahead of print]

Zhang YJ, Bonaparte RS, Patel D, Stein DA, Iversen PL. Blockade of viral interleukin-6 expression of Kaposi's sarcoma-associated herpesvirus. Mol Cancer Ther. 2008 Mar;7(3):712-20.

Stone JK, Rijnbrand R, Stein DA, Ma Y, Yang Y, Iversen PL, Andino R. A morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus. Antimicrob Agents Chemother. 2008 Jun;52(6):1970-81. Epub 2008 Mar 17.

Gabriel G, Nordmann A, Stein DA, Iversen PL, Klenk HD. Morpholino oligomers targeting the PB1 and NP genes enhance the survival of mice infected with highly pathogenic influenza A H7N7 virus. J Gen Virol. 2008 Apr;89(Pt 4):939-48.

Vagnozzi A, Stein DA, Iversen PL, Rieder E. Inhibition of foot-and-mouth disease virus in cell cultures with antisense Morpholino oligomers. J Virol. 2007 Aug 29; [Epub ahead of print]

Marshall NB, Oda SK, London CA, Moulton HM, Iversen PL, Kerkvliet NI, Mourich DV. Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing. J Immunol Methods. 2007 Jul 26; [Epub ahead of print]

Wu RP, Youngblood DS, Hassinger JN, Lovejoy CE, Nelson MH, Iversen PL, Moulton HM. Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity. Nucleic Acids Res. 2007 Aug 1; [Epub ahead of print]

Moulton HM, Fletcher S, Neuman BW, McClorey G, Stein DA, Abes S, Wilton SD, Buchmeier MJ, Lebleu B, Iversen PL. Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo. Biochem Soc Trans. 2007 Aug;35(Pt 4):826-8.

Amantana A, Moulton HM, Cate ML, Reddy MT, Whitehead T, Hassinger JN, Youngblood DS, Iversen PL. Pharmacokinetics, Biodistribution, Stability and Toxicity of a Cell-Penetrating Peptide-Morpholino Oligomer Conjugate. Bioconjug Chem. 2007 Jun 21; [Epub ahead of print]

Fletcher S, Honeyman K, Fall AM, Harding PL, Johnsen RD, Steinhaus JP, Moulton HM, Iversen PL, Wilton SD. Morpholino Oligomer-Mediated Exon Skipping Averts the Onset of Dystrophic Pathology in the mdx Mouse. Mol Ther. 2007 Jun 19; [Epub ahead of print]

Deas TS, Bennett CJ, Jones SA, Tilgner M, Ren P, Behr MJ, Stein DA, Iversen PL, Kramer LD, Bernard KA, Shi PY. In vitro resistance selection and in vivo efficacy of morpholino oligomers against West Nile virus. Antimicrob Agents Chemother. 2007 May 7; [Epub ahead of print] {in-vivo mouse data}

Burrer R, Neuman BW, Ting JPC, Stein DA, Moulton HM, Iversen PL, Kuhn P, Buchmeier MJ. Antiviral effects of antisense morpholino oligomers in murine coronavirus infection models. J. Virol. 2007 Epub 2007 Mar 5.{in-vivo mouse data}

Yuan J, Stein DA, Lim T, Qiu D, Coughlin S, Liu Z, Wang Y, Bouch R, Moulton HM, Iversen PL, Yang D. Inhibition of Coxsackievirus B3 in Cell-cultures and in Mice by Peptide-Conjugated Morpholino Oligomers Targeting the IRES. J Virol. 2006 Sep 20; [Epub ahead of print]

Abes S, Moulton HM, Clair P, Prevot P, Youngblood DS, Wu RP, Iversen PL, Lebleu B. Vectorization of morpholino oligomers by the (R-Ahx-R)(4) peptide allows efficient splicing correction in the absence of endosomolytic agents. J Control Release. 2006 Sep 30; [Epub ahead of print] {nice mechanism study, also a good place to start on how to conjugate}

Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM. Stability of cell-penetrating Peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 2007 Jan-Feb;18(1):50-60.

McClorey G, Fall AM, Moulton HM, Iversen PL, Rasko JE, Ryan M, Fletcher S, Wilton SD. Induced dystrophin exon skipping in human muscle explants. Neuromuscul Disord. 2006 Oct;16(9-10):583-90. Epub 2006 Aug 21 {application paper, little background on the conjugates}

McClorey G, Moulton HM, Iversen PL, Fletcher S, Wilton SD. Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD. Gene Ther. 2006 Oct;13(19):1373-81. Epub 2006 May 25 {application paper, little background on the conjugates}

Deas TS, Binduga-Gajewska I, Tilgner M, Ren P, Stein DA, Moulton HM, Iversen PL, Kauffman EB, Kramer LD, Shi PY. Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication. J Virol. 2005 Apr;79(8):4599-609 {application paper, little background on the conjugates}

Nelson MH, Stein DA, Kroeker AD, Hatlevig SA, Iversen PL, Moulton HM. Arginine-rich peptide conjugation to morpholino oligomers: effects on antisense activity and specificity. Bioconjug Chem. 2005 Jul-Aug;16(4):959-66. {Nice paper specifically on the conjugates}

Moulton HM, Moulton JD. Arginine-rich cell-penetrating peptides with uncharged antisense oligomers. Drug Discov Today. 2004 Oct 15;9(20):870. {argument for the effectiveness of the Morpholino-peptide system}

Moulton HM, Nelson MH, Hatlevig SA, Reddy MT, Iversen PL. Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. Bioconjug Chem. 2004 Mar-Apr;15(2):290-9. {Nice paper specifically on the conjugates}

Moulton HM, Moulton JD. Peptide-assisted delivery of steric-blocking antisense oligomers. Curr Opin Mol Ther. 2003 Apr;5(2):123-32. {review}

Moulton HM, Hase MC, Smith KM, Iversen PL. HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev. 2003 Feb;13(1):31-43. {early exploration with less-optimal peptide}
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Postby MrMistery » Fri Nov 07, 2008 6:53 pm

Do you mean it is possible to knockout a gene in an adult human?
It may be possible in knockout in an adult C. elegans, but its not quite the same thing.
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Re: is it possible to knockdown a gene in a living adult now

Postby jonmoulton » Fri Nov 07, 2008 7:12 pm

I expect that AVI BioPharma http://www.avibio.com will start systemic clinical trials of their peptide-conjugated oligos targeting dystrophin for treatment of Duchenne muscular dystrophy within a year. Their trials with bare Morpholinos have been ongoing in London.
http://clinicaltrials.gov/ct2/show/NCT00159250

Of course, without clinical trial data we cannot make certain statements about the efficacy of the Morpholino-peptide conjugates in human systems. But they work in other mammals, as discussed in the publications I cited previously.

Another option for in-vivo delivery of antisense are the Vivo-Morpholinos, also tested successfully in mammals. These are Morpholinos covalently conjugated to octaguanidinium dendrimers.

Li YF, Morcos PA. Design and Synthesis of Dendritic Molecular Transporter that Achieves Efficient in Vivo Delivery of Morpholino Antisense Oligo. Bioconjug Chem. 2008 Jul;19(7):1464-70. Epub 2008 Jun 20.

I didn't cite these in previous list because they are newer and still have a scant publication record. There are some systemic mouse data in the Bioconjugate Chemistry paper. There will be another paper, focused more on the biology, in the December 2008 Biotechniques.
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Postby MrMistery » Sat Nov 08, 2008 1:18 am

neat stuff.. thanks for the info
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Re: is it possible to knockdown a gene in a living adult now

Postby jonmoulton » Tue Nov 18, 2008 6:05 pm

Here we go -- the first clinical trial with a Morpholino-peptide conjugate.

http://www.bioresearchonline.com/article.mvc/AVI-BioPharma-Announces-Initiation-Of-0001

Start of press release:

AVI BioPharma Announces Initiation Of Clinical Trial Of Next Generation Drug Eluting Stent By Partner Global Therapeutics

November 11, 2008

AVI BioPharma, Inc., a developer of RNA-based drugs, recently announced that its partner Global Therapeutics, a Cook Medical company, has initiated the world's first clinical trial of a drug eluting stent that uses a PPMO (peptide-conjugated morpholino phosphorodiamidate oligomer)-based RNA therapeutic agent aimed at silencing C-MYC, one of the genes responsible for causing arteries to reclose after stenting (restenosis).

<snip>

------------------------------------------

http://www.cookmedical.com/newsDetail.do?id=3910

Another from Cook Medical about the same trial:

Global Therapeutics Initiates Trial of Next Generation Drug-Eluting Stent Using RNA Therapeutic Agent to Treat Coronary Artery Disease

November 10, 2008

Broomfield, Colo., November 10, 2008 — Global Therapeutics, a leading developer of innovative solutions for the cardiology market, today announced initiation of the world's first clinical trial of a drug eluting stent that uses an antisense RNA therapeutics agent aimed at silencing one of the genes (c-myc) responsible for causing arteries to reclose after stenting (restenosis).

<snip>
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Postby Cat » Sun Nov 30, 2008 8:40 pm

Gene knock out and RNA therapy are different things. The answer to the question is yes, it is possible, but not practical. Site directed DNA mutagenesis is difficult and near impossible to achieve that is the primary reason for using alternative methods. However, random gene knockout is simple - however impractical that might be.
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Postby jonmoulton » Mon Dec 01, 2008 5:03 pm

True of knockouts, Cat. The original question was about knockdowns, not knockouts.

is it possible to knockdown a gene in a living adult now?
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Re: is it possible to knockdown a gene in a living adult now

Postby jonmoulton » Fri Dec 05, 2008 8:41 pm

NewsFocus article on antisense exon-skipping therapy for Duchenne muscular dystrophy:

Hopping to a better protein. Science 5 Dec 2008, v. 322, p.1454-5

Audio supplement: http://www.sciencemag.org/content/vol322/issue5907/images/data/1454/DC1/1454.mp3

"Clinical trials are under way to test an innovative use of antisense technology to stem paralysis in Duchenne muscular dystrophy"

This article give progress updates, quotes from scientists involved in developing exon-skipping therapeutics, and describes how exon skipping works. They discuss the regulatory problems of having many different sequences needed to treat many different people each with their own unique mutation.

One complaint: the Science article says that "Qi Long Lu, a pathologist at Carolinas Medical Center in Charlotte, North Carolina, has added a peptide rich in the amino acid arginine to the morpholino antisense." Sorry, Science, you got that one wrong. That peptide was developed and added by my wife, Hong Moulton of AVI BioPharma.

Qi Long Lu received the peptide-conjugated Morpholino from Hong and conducted his excellent studies in mice: Wu B, Moulton HM, Iversen PL, Jiang J, Li J, Li J, Spurney CF, Sali A, Guerron AD, Nagaraju K, Doran T, Lu P, Xiao X, Lu QL. Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci U S A. 2008 Sep 19;105(39):14814-9.

----------------------------------------------

Clinical trial link, US Gov't clinical trials website:
http://clinicaltrials.gov/ct2/show/NCT00159250?term=morpholino+dmd&rank=1

Description of the trial from Duchenne Connect:
https://www.duchenneconnect.org/index.php?option=com_content&task=view&id=172&Itemid=147&trial_id=NCT00159250

Manufacturers of the Morpholino oligos used in the clinical trial:
http://www.avibio.com

----------------------------------------------

I'd like to add some comments about the current realities of antisense drug development for genetic disease with DMD as the model.

Antisense therapeutics are the obvious intersection of therapeutics with sequence data. As more individual sequences become available, specific mutations causing disease will sometimes be found which are amenable to modification with therapeutic antisense. However, the current regulatory system of the USA or Europe will not allow development and use of sequence-specific therapeutic molecules on an individualized basis.

Eric Hoffman, a leader in DMD research, published an interesting article relevant to this problem. He proposes that significant change in drug regulation will be needed to allow development of personalized antisense drugs and treatment of the broadest range of DMD-causing mutations with antisense.

Hoffman EP. Skipping toward personalized molecular medicine. N Engl J Med. 2007 Dec 27;357(26):2719-22.

Consider the problem of the cost of clinical trials. Under the current regulatory system, there will hopefully be a few oligo sequences that can treat large enough populations of DMD patients that there is a chance the clinical trials could be paid off by drug revenue. However, for many patients these large-population oligo sequences will not be the right fit for their specific mutation. While in principle an oligo could be made to treat their mutation, no drug company will fund the crushing expense of a clinical trial without hope of recovering the investment. We have the technology that could help them, but the intersection of the regulatory and economic forces will leave them suffering -- unless we muster the will to change the regulations or the economic landscape. See Hoffman's paper for a more complete discussion of this regulatory-economic trap.
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Postby jonmoulton » Tue Dec 09, 2008 5:56 pm

A new open-access paper describes delivery of Vivo-Morpholinos in cultures, EGFP splice-reporter mice and mdx mice, including tissue-specific delivery measurements by RT-PCR and measurement of dystrophin exon-skipping by RT-PCR and immunohistochemistry:

Paul A. Morcos, Yongfu Li, and Shan Jiang. Vivo-Morpholinos: A non-peptide transporter delivers Morpholinos into a wide array of mouse tissues. BioTechniques. 2008 Dec;45(6):616-26.

http://www.biotechniques.com/default.as ... &id=113005
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