Researching Multi Gene Diseases & Gene Therapy

[gravenewworld]

Over the past 20 years, academia and industry has moved heavily towards research focused upon gene therapy. One of the problems with this research is that it only seems to study diseases that are based off of a single malfunctioning gene. Many of the biggest health problems like cardiovascular disease, arthritis, etc. are believed to be related to multiple gene failures. So my question is this. How exactly could we ever come up with a technology to transfect a cell with multiple genes? Even if you get your little snippet of DNA or other type of nucleic acid into the cell, how could you ever control each gene you are delivering so that it gets directed to the proper chromosome if you are trying to treat a multigene related disease that are associated with genes across multiple chromosomes? It seems like gene therapy is severely limited based off the complex and multifactorial aspects of many diseases. Does anyone know of any research out there currently being done that addresses the issue of trying to treat multigene related disease with gene therapy? I mean it is nice to be able to treat a disease related to a single gene like sickle-cell anemia, but many other major disorders are much more complex at the chromosomal/dna level.

 

[zhermes]

Every point you bring up is spot on. But you're missing the key feature: gene therapy is being researched (almost entirely) for single-gene defects. There are plenty of single cell issues that make the approach entirely worth while.

Sickle-cell, while the classic example, actually gets a little complicated; but consider some other examples of diseases which might be treatable with gene therapy:
HIV, many types of cancer, Huntington's disease, muscular dystrophy, etc etc.
Even if one of these was the only application of gene therapy---it would be worth-while, and incredibly profitable (except for HIV, maybe).

No generic types of treatments can be used to treat everything.

At the same time, I have no doubt that at some point in the future multi-gene, gene-therapies will become used and useful. Just not yet.

 

[gravenewworld]

Every point you bring up is spot on. But you're missing the key feature: gene therapy is being researched (almost entirely) for single-gene defects. There are plenty of single cell issues that make the approach entirely worth while.

Sickle-cell, while the classic example, actually gets a little complicated; but consider some other examples of diseases which might be treatable with gene therapy:
HIV, many types of cancer, Huntington's disease, muscular dystrophy, etc etc.
Even if one of these was the only application of gene therapy---it would be worth-while, and incredibly profitable (except for HIV, maybe).

No generic types of treatments can be used to treat everything.

At the same time, I have no doubt that at some point in the future multi-gene, gene-therapies will become used and useful. Just not yet.

Worthwhile I definitely agree with you. Especially if you are at an academic institution. But of course industry cares first about economics and profit. I mean is there really that much money for treating a rare disease like muscular dystrophy or huntington's if the occurence of those diseases is quite low amongst the general population? I feel like if it doesn't have at least $200+ million potential, a disease isn't going to attract a lot of industrial research.


As for cancer, wouldn't it be much more prudent to use say a single drug that can kill multiple different types of cancer, but rather than focusing on specific cancer genes, exploit a common characteristic that many types of cancers have in common so that you can deliver the cancer killing agent specifically to cancer cells? That way you don't have to research which specific gene is causing cancer for every individual type of cancer, come up with the repaired dna sequence, and then figure out exactly to get the gene inside the cancer cell.


I'm not an expert at all when it comes to these topics, these are just some random thoughts I have before going to grad school I'd like to address before possibly delving into a project aimed at gene delivery for the next 6 years.

 

[Andy Resnick]

As for cancer, wouldn't it be much more prudent to use say a single drug that can kill multiple different types of cancer, but rather than focusing on specific cancer genes, exploit a common characteristic that many types of cancers have in common so that you can deliver the cancer killing agent specifically to cancer cells? That way you don't have to research which specific gene is causing cancer for every individual type of cancer, come up with the repaired dna sequence, and then figure out exactly to get the gene inside the cancer cell.

That's essentially what chemotherapy does- it targets cell surface receptors of rapidly-dividing cells (CD21, CD40, etc). That's why perfectly healthy cells that divide rapidly (hair, gut epithelia, etc) are also killed during chemotherapy.

To your OP question tho- yes, there are many diseases that result from whole families of bad genes- Bardet-Biedel syndrome, for example.

 

[zhermes]

You make a good argument. But, personally, I still think its incredibly worth it (even outside of an academic stand-point, which I would fall back on).

I mean is there really that much money for treating a rare disease like muscular dystrophy or huntington's if the occurence of those diseases is quite low amongst the general population? I feel like if it doesn't have at least $200+ million potential, a disease isn't going to attract a lot of industrial research.

Yeah, maybe my "one would be enough" statement was a little too grandiose---but I think there are plenty of 1-gene diseases for it to be highly profitable. Single treatments (especially for exotic diseases) can cost tens to hundreds of thousands of dollars.

As for cancer, wouldn't it be much more prudent to use say a single drug that can kill multiple different types of cancer...That way you don't have to research which specific gene is causing cancer for every individual type of cancer...

Theoretically yes. But generally that doesn't really exist. Those drugs have moderate efficacy, and very high collateral damage. Those drugs are almost always treatments, where-as gene therapy (in combination with those drugs) could be a cure. There are already many known genes that contribute to cancer, and knowledge of those genes is important for general drug development and biological understanding (i.e. not *only* useful for gene therapy). Most cancer's require many different problems in the cell to develop; to one extent this reinforces your standpoint (that you need multi-gene treatment), but at the same time it suggests that repairing some single genes (e.g. p53) could be enough to cure it.

 

[bobze]

Worthwhile I definitely agree with you. Especially if you are at an academic institution. But of course industry cares first about economics and profit. I mean is there really that much money for treating a rare disease like muscular dystrophy or huntington's if the occurence of those diseases is quite low amongst the general population? I feel like if it doesn't have at least $200+ million potential, a disease isn't going to attract a lot of industrial research.


As for cancer, wouldn't it be much more prudent to use say a single drug that can kill multiple different types of cancer, but rather than focusing on specific cancer genes, exploit a common characteristic that many types of cancers have in common so that you can deliver the cancer killing agent specifically to cancer cells? That way you don't have to research which specific gene is causing cancer for every individual type of cancer, come up with the repaired dna sequence, and then figure out exactly to get the gene inside the cancer cell.


I'm not an expert at all when it comes to these topics, these are just some random thoughts I have before going to grad school I'd like to address before possibly delving into a project aimed at gene delivery for the next 6 years.

Cancer genetics gets tricky to "cure" because each cancer is really a unique beast. Certainly certain genes like Rb, p53, certain tumor suppressor miRNAs etc go bad, but once point of genomic instability is reached, there are many, many genetic changes to the cell lineage.

To your OP, its even more complicated than just multiple genes. It often involves (like cardiovascular disease) environmental factors. We call these 'multifactorial' disease. Treatment for these diseases will always require a multiple angle approach because the factors (different genes, gene dosages, environmental factors, etc) cover such a wide range of things.

Single targeted gene therapy is certainly a worthwhile "industrial" investment. Consider something like cystic fibrosis (CF) which we are experimentally treating with gene therapy now in 2 ways. Adenoviruses are being used to transfect respiratory epithelium with functional chloride channels (the gene product of the CF gene, CTFR) and also through plasmid introduction via liposomes.

The big problem with trasfection of cell lines for gene therapy is that where the functional gene is inserted into the genome is really random. If you're lucky, the functional gene and promoter will insert in a good spot--If you're unlucky it may just wreck one of those essential tumor suppressor genes; like p53 (which just 1 'bad' copy is enough to cause cancerous growth states).

Plasmid introduction via liposomes seems to the best current alternative, but (good for the drug companies, I guess) require constant treatment and you can eventually develop an immune reaction to the liposomes themselves. Certainly an interesting topic and with the sequencing of the human genome done, increasing computer technology and bioinformatics and our understanding of molecular genetics it certainly appears to be a promising future (for patients, "academics" and "industry").

 

[gravenewworld]

Cancer genetics gets tricky to "cure" because each cancer is really a unique beast. Certainly certain genes like Rb, p53, certain tumor suppressor miRNAs etc go bad, but once point of genomic instability is reached, there are many, many genetic changes to the cell lineage.

To your OP, its even more complicated than just multiple genes. It often involves (like cardiovascular disease) environmental factors. We call these 'multifactorial' disease. Treatment for these diseases will always require a multiple angle approach because the factors (different genes, gene dosages, environmental factors, etc) cover such a wide range of things.

Single targeted gene therapy is certainly a worthwhile "industrial" investment. Consider something like cystic fibrosis (CF) which we are experimentally treating with gene therapy now in 2 ways. Adenoviruses are being used to transfect respiratory epithelium with functional chloride channels (the gene product of the CF gene, CTFR) and also through plasmid introduction via liposomes.

The big problem with trasfection of cell lines for gene therapy is that where the functional gene is inserted into the genome is really random. If you're lucky, the functional gene and promoter will insert in a good spot--If you're unlucky it may just wreck one of those essential tumor suppressor genes; like p53 (which just 1 'bad' copy is enough to cause cancerous growth states).

Plasmid introduction via liposomes seems to the best current alternative, but (good for the drug companies, I guess) require constant treatment and you can eventually develop an immune reaction to the liposomes themselves. Certainly an interesting topic and with the sequencing of the human genome done, increasing computer technology and bioinformatics and our understanding of molecular genetics it certainly appears to be a promising future (for patients, "academics" and "industry").

This was going to be my next question. I guess it wouldn't really matter if it were a multi gene or single gene related disease, I mean what happens to an exogenously delivered gene once it is inside the nucleus? I mean does the cell have some sort of machinery that it can take a little snippet of DNA and incorporate it at the exact precise location on the chromosome? I'd imagine that if you deliver a piece of DNA in the wrong spot on a chromosome you'd cause all sorts of other problems like frame shift mutations. All the work being done now seems to be entire focused on just trying to deliver DNA with cell specificity. What happens at the subcellular level? Is there even any research being done on manipulating the cell machinery so that you can have guided incorporation of a gene into the right location of the genome?

 

[Andy Resnick]

This was going to be my next question. I guess it wouldn't really matter if it were a multi gene or single gene related disease, I mean what happens to an exogenously delivered gene once it is inside the nucleus? I mean does the cell have some sort of machinery that it can take a little snippet of DNA and incorporate it at the exact precise location on the chromosome? I'd imagine that if you deliver a piece of DNA in the wrong spot on a chromosome you'd cause all sorts of other problems like frame shift mutations. All the work being done now seems to be entire focused on just trying to deliver DNA with cell specificity. What happens at the subcellular level? Is there even any research being done on manipulating the cell machinery so that you can have guided incorporation of a gene into the right location of the genome?

That's an interesting question, and I can only give an approximate answer:

The genes that I insert into my cells are not isolated genes (for say, GFP-tagged proteins), but also contain "promoter sequences", "antibiotic resistance genes", and a few other things. What I mean is that a functional gene is not just an isolated snippet of DNA- there is also a promoter region (and possibly an inhibitor region) that controls how much that gene is read and expressed.

http://en.wikipedia.org/wiki/TATA_box

So, the specific location in a chromosome (or selection of a specific chromosome) does not appear to be that important- but altering the promoter sequence can change a gene from being silenced to being over-expressed.

 

[bobze]

This was going to be my next question. I guess it wouldn't really matter if it were a multi gene or single gene related disease, I mean what happens to an exogenously delivered gene once it is inside the nucleus? I mean does the cell have some sort of machinery that it can take a little snippet of DNA and incorporate it at the exact precise location on the chromosome? I'd imagine that if you deliver a piece of DNA in the wrong spot on a chromosome you'd cause all sorts of other problems like frame shift mutations. All the work being done now seems to be entire focused on just trying to deliver DNA with cell specificity. What happens at the subcellular level? Is there even any research being done on manipulating the cell machinery so that you can have guided incorporation of a gene into the right location of the genome?

Yes, there is certainly research being done on making gene insertion less dangerous.

As Andy points out, we provide all the necessary "stuff" with the gene when it is delivered. For researchers and some gene therapies, this often means using a viral promoter sequence--As they tend to get good "active" results for the genes you want to be expressed.

For long term viability of gene therapy though ("real" gene therapy) then we'd need to gene into a cell, where it is subject to all those "normal" gene regulatory regions. This gets complex and is slow research because we have found that gene regulation is much, much more complicated than anyone thought. Its complicated in prokaryotes, but gets to a whole new level of complexity when you start looking at things like mammals.

Plasmids seem a good alternative to not "wrecking" the genome at the moment, but the problem with them, is your cells have little "policing" agents which like to, over time, kick the plasmids out of the party. This means, that gene therapy with plasmids would need to be an ongoing treatment. Also, as I mentioned above, the way we deliver plasmids for gene therapy (liposomes) can cause an immune reaction against them. Which renders the therapy rather mute.

That's an interesting question, and I can only give an approximate answer:

The genes that I insert into my cells are not isolated genes (for say, GFP-tagged proteins), but also contain "promoter sequences", "antibiotic resistance genes", and a few other things. What I mean is that a functional gene is not just an isolated snippet of DNA- there is also a promoter region (and possibly an inhibitor region) that controls how much that gene is read and expressed.

http://en.wikipedia.org/wiki/TATA_box

So, the specific location in a chromosome (or selection of a specific chromosome) does not appear to be that important- but altering the promoter sequence can change a gene from being silenced to being over-expressed.

Just to clarify for the OP Andy, gene insertion in vitro (in the lab) and in vivo (like we'd need for clinical usage) are really different things.

I've transfected lots of immortal cell lines and bacteria. When you use something like a reverse transcriptase to do the transfection, you do inevitably wreck some chromosomes and cause some cells to die. The difference, in vitro, is you are dealing with log scales of bacterial or transformed cells and loosing a few is small fish.

Clinically then; it becomes a problem because while we can certainly think of people as "giant petri dishes of cells", the function of the organism does depend on the function of it's cells. Wrecking some, may not be well tolerated by the organism or lead to undesirable clinical outcomes (why does everything always seem easier in lab )

For example, the some of the first clinical applications of gene therapy where in individuals lacking Adenosine deaminase, also known as severe combined immunodeficiency (SCID) aka: "bubble boy".

Functional Adenosine deaminase was RT'd into cell lines in the patients and it seemed at first, to work out great. The problem, which pops up a couple of years down the road--is they start getting cancers.

Now, to me, this is one of those "risk/reward" scenarios where the reward, certainly outweighs the risk. We can treat those cancers and prolong life--However, you cannot live without an immune system. Case in point, people with SCID rarely life past their second birthday.

However, looking at someone with CF--Who is likely to live into their 40's with current treatments, for aggressive gene therapy the risk of doing it at an early age certainly outweighs the reward (hence, we've moved toward less aggressive gene therapies such as plasmid introduction through liposomes).

For gene therapy to really be successful in the long term, we are going to need a way which provides us with some sort of control regarding the introduction of genes into cell lines--Otherwise well always have that pink elephant (what did we wreck?) in the room.

 

[Andy Resnick]

Just to clarify for the OP Andy, gene insertion in vitro (in the lab) and in vivo (like we'd need for clinical usage) are really different things.

Absolutely- sorry I didn't make that clear.