Exploring the Potential of Knockout Technology in Human Cells: An Overview

[bioquest]

"knockout" technology

Does "knockout" technology work not work in human cells at this time, leaving only the option of RNA interference? I thought you could use knockout technology in human cells

 

[BoomBoom]

A gene "knockout" would be an unethical thing to do in a human. That is why it is generally done with mice. You are basically creating a new life-form that has a certain gene altered through an insertion making it non-functional or absent. I suppose you could remove genetic material and "cut-out" a gene (probably with a specific restriction enzyme) then re-insert it into a new vacant egg and start a new life (similar to how they clone animals).

RNA interference can be done with anti-sense RNA which is just a complimentary strand of RNA to the RNA you want to inhibit so that it binds to the target RNA and it cannot continue through with the protein production process. However, if the gene if still expressing, then this is just a temporary inhibition.

The jist I get from your various threads is that you want to know if there is some way to "delete" a gene in a living human, and I believe the answer is NO.

 

[bioquest]

So if a human wanted their genes altered (they do gene therapy for medical purposes right?) even if it was done for medical purposes and was legal, it would be physically impossible to delete a gene in them? if they weren't an embryo?

Could you only remove genetic material and "cut-out" a gene in an organism that wasn't already an adult?

 

[BoomBoom]

So if a human wanted their genes altered (they do gene therapy for medical purposes right?) even if it was done for medical purposes and was legal, it would be physically impossible to delete a gene in them? if they weren't an embryo?

Could you only remove genetic material and "cut-out" a gene in an organism that wasn't already an adult?

Yeah, as far as I know you cannot "cut-out" a gene from a living being.

Some gene therapy does go on and they are still learning about this new field. As far as I know, most succesfull gene therapy is done with blood cell lines and components. Since a bone marrow transplant can actually eventually replace defective genes with working ones due to the nature of bone marrow itself (producing new blood cells).

I think you run into some difficulty though with other tissue types. You could probably deliver modified genetic material to targeted tissues with some viruses and retro-viruses, but it would just add to the genetic material that is there already, and you'd want to be careful to deliver this ONLY to the targeted tissue.

I don't really know, but I do know that this field of research is on-going and growing and who knows what the future holds?

 

[BoomBoom]

Probably the best method, if you wanted to inhibit a specific gene from expressing, might be to design some sort of inhibitor that was designed to bind to that gene's specific binding site to stop the polymerase from binding to that gene.

Of course delivery is a whole other ball of wax...

 

[Andy Resnick]

Does "knockout" technology work not work in human cells at this time, leaving only the option of RNA interference? I thought you could use knockout technology in human cells

Creating a knockout mouse takes 1-2 years, and is quite involved. It involves controlled breeding to create a stable line in addition to manipulating the ova. Humans would not be a good candidate for this type of manipulation.

Creating a transfected cell line is substantially easier, but still requires a cell line as opposed to primary cells. I created a stabily transfected cell line in about 2 weeks.

There are techniques like RNAi, morpholino, and Cre/Lox which can knockdown specific genes (possibly in a tissue-specific manner), but I've never used those methods.

The gene therapy trials I know of on humans have been RNAi, and the results have been underwhelming AFAIK.

 

[bioquest]

Could a transfected cell line be used to introduce and spread a virus, in an organism like a mouse?

 

[Andy Resnick]

Why not just introduce the virus directly? Lentivirus is a commonly used transfection vector.

 

[JonMoulton]

An alternative method for gene knockdown in humans is Morpholino antisense (thanks, Andy). A knockdown is temporary suppression of gene expression, similar to the effect of RNAi (though Morpholinos have some advantages over siRNA: http://www.gene-tools.com/files/Summerton2007siRNAcompare.pdf ). The company AVI BioPharma has been conducting clinical trials with Morpholino oligos (www.avibio.com). Morpholinos are widely used in developmental biology research, especially in zebrafish.

Morpholinos can knockdown protein expression by interrupting the path of a small ribosomal subunit from the 5'-cap to the start codon, preventing formation of a mature ribosome (http://www.gene-tools.com/files/bba%20review.pdf ). They are also used to redirect pre-mRNA splicing by blocking snRNP binding sites (http://www.gene-tools.com/files/draper_etal.pdf ). Finally, Morpholinos are being used to block miRNA maturation and activity (Ref 1). Effective delivery into the cells of adult organisms has been achieved by conjugation with arginine-rich cell-penetrating peptides (Ref 2,3,4) and by octaguanidinium moieties (www.gene-tools.com/vivomorpholinos).

The modification of splicing is a form of gene deletion; while a splice-targeted Morpholino directs the removal of a single exon, if that exon has a number of bases not evenly divisible by three then the downstream exon sequences will be frameshifted, generally leading to production of a nonfunctional protein.

References
(1) Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development. PLoS Biol. 2007 Jul 24;5(8):e203 [Epub ahead of print]

(2) 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]

(3) 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.

(4) 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]