Genetic engineering at home

[rafael diaz]

I want to incorporate certain genes from a certain animal into myself. I was thinking of using a retrovirus with the gene. Would this work? Where would I be able to find the resources I need? Is there a easier/more possible way of accomplishing this? Thanks in advance

[Brian Degger]

You might want to ask a biosafety expert first.

Legalities of this idea are problematical, ethics are questionable..biosafety issues arw imediately applicable and I would guess anyone who had access to the technology wouldn't be granting access.
How would you prevent it infecting others? Could you cause yourself cancer?

On 30 Dec 2013 15:06, "rafael diaz" <raphae...@gmail.com> wrote:

I want to incorporate certain genes from a certain animal into myself. I was thinking of using a retrovirus with the gene. Would this work? Where would I be able to find the resources I need? Is there a easier/more possible way of accomplishing this? Thanks in advance

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[Mega [Andreas Stuermer]]

If that is legal in your country:

How do you want to deliver the gene into all of your cells? Would be easier to engineer sperm cells (but AFAIK, that is legal quite nowhere). Let aside ethical aspects.

You perhaps can engineer e.g. your eyes to see infrared if you put a replication-deficient virus on them that introduces somke kind of pigment that senses infrared.

Or want something like repoxygen?

[Mega [Andreas Stuermer]]

Just for your info, not that I say this would be nice at all,

> How would you prevent it infecting others?

Replication defective viruses. But that also prevents it from infecting all of your cells that it hadn't had contact to.

> Could you cause yourself cancer?

Adeno-associated virus does often not integrate. And if it does, it targets a specific locus.

Do you intend to cure a disease in you? Or just enhance your body - transhumanism

[Patrik D'haeseleer]

This is not something I would recommend trying, unless you know really well what you're doing, and what the possible consequences can be - in which case you wouldn't be asking this question here to begin with.

Possible side effects may include inflammation, cancer, toxic shock, gangrene, autoimmune disease, generating a dangerous new communicable disease, or disintegrating a la The Fly minus the super powers - in roughly decreasing order of likelihood.

If anyone really wanted to pursue this, I would strongly suggest NOT starting with something as ambitious as antiretroviral gene therapy. I bet it's only a matter of time before someone tries to give themselves a GFP tattoo with a gene gun though...

[Ravasz]

As the others explained, getting an animal's gene into your body is not a possibility today. I'm not even sure if making this accessible would be a good idea...

If you are interested in genetic engineering, then perhaps you could transfer a gene from one bacteria species to the other. This is much easier to accomplish, and could lead to very interesting results while staying mostly harmless.

[Paul Schroeer-Hannemann]

And just what do you think this would accomplish?  Give you fur?  Supersonic hearing?  Claws?  A dog's nose?  

Genes aren't Legos, they're recipes for proteins.  And adding some protein to your mix all willy-nilly like that can have disastrous consequences.

[Nathan McCorkle]

On Dec 30, 2013 10:10 PM, "Patrik D&apos;haeseleer" <pat...@gmail.com> wrote:
>I bet it's only a matter of time before someone tries to give themselves a GFP tattoo with a gene gun though...
>

That's already started happening, at least academically:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855251/

[Cathal Garvey (Phone)]

Also, I have seen a phonecam shot of an alleged GFP "tattoo" where someone jabbed themselves with an Adenoviral gfp vector. I say "Alleged" because I can't imagine that working for more than an hour or so before going necrotic from immune rejection.

--
Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Nathan McCorkle]

There are also experiments where they placed naked GFP with a promoter
in the nasal cavity of a mouse, later they saw the liver fluorescing.
The reference evades me now.

This Jove video also shows the tattooing of a mouse with GFP, but they
seem to have sectioned the tissue later, so it doesn't mention
necrosis, etc...
http://www.jove.com/video/50032/skin-tattooing-as-a-novel-approach-for-dna-vaccine-delivery

full video:
http://ecsource.jove.com/CDNSource/50032_ASV_082612_Web.mov

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-Nathan

[Mega [Andreas Stuermer]]

If the person would have chosen adeno-asssociated virus instead of adenovirus, that shouldn't led to necrosis and should (in theory) be perfectly safe, right?

Only problem will be, skin cells renew very often. After, say, 4 weeks there will hardly be GFP marked cells anymore because they got replaced by stem cells. Or are the stem cells in reach to be transfected too? Then it would be a permanent tatoo.

[Pieter]

I was shown the same picture, and share your skepticism. It seems pretty unlikely, and not documented elsewhere...

[Nathan McCorkle]

Realistically though, would the immune reaction be more than something
like a bee-sting causes? GFP isn't an enzyme like bee-toxin is, just a
non-self protein. Maybe a more similar allergenic situation would be a
skin-allergen prick test.
http://en.wikipedia.org/wiki/Skin_allergy_test

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[Cathal Garvey]

Makes no difference what vector you use. AAV won't trigger as much of an
immune response to the *viral vector*, but once cells start expressing a
foreign protein like GFP, the body will start attacking them as if they
are infected with a virus, no matter what method you use to get the DNA
in there.

The few exceptions (loosely) are things like Neurons and Muscles, which
the body tries to avoid attacking even for immunological reasons..but
which would be even worse than skin if the body *does* attack anyway. So
don't go there.

My expectation in general for gene therapy is that if the introduced
protein is entirely foreign (and not, for example, a similar but
better-functionality version of a natural human protein), you should
expect the immune system to destroy transformed cells sometime after
transformation. Whether this manifests as a bad itch and redness, or
loss of a patch of skin to infected necrotic tissue, is just a matter of
scale!

Your eyes, if you want infrared-vision, are actually a potentially safe
place to play, because the immune system *might* give eyes a free pass.
But then you're risking eye cancer, and the infrared vision probably
won't happen anyway, because vision is about more than just receptors;
you also need your optic nerve to deliver your receptor signal to the
brain in a way that's distinguishable from other colours, and I'm not
convinced that's within our technology right now unless engineering
germline/embryos (eg. before their brain learns to distinguish signals
and colours).

All of which is beside the basic point that you shouldn't hack your own
genes. We have the technology but not the skill; nobody sensible should
be offering gene therapy for anything but important medical applications
like cancer therapy or for vision loss. It's not worth the risk of
cancer or necrosis just to have a GFP tattoo when you could just get
fluorescent tattoo dye and get the same effect (and probably better
quantum yield!).

On 31/12/13 22:27, Mega [Andreas Stuermer] wrote:
> If the person would have chosen adeno-asssociated virus instead of
> adenovirus, that shouldn't led to necrosis and should (in theory) be
> perfectly safe, right?
>
> Only problem will be, skin cells renew very often. After, say, 4 weeks
> there will hardly be GFP marked cells anymore because they got replaced by
> stem cells. Or are the stem cells in reach to be transfected too? Then it
> would be a permanent tatoo.
>
>
>
>
> On Tuesday, December 31, 2013 8:00:38 PM UTC+1, Cathal Garvey (Phone) wrote:
>>
>> Also, I have seen a phonecam shot of an alleged GFP "tattoo" where someone
>> jabbed themselves with an Adenoviral gfp vector. I say "Alleged" because I
>> can't imagine that working for more than an hour or so before going
>> necrotic from immune rejection.
>>

>> Nathan McCorkle <nmz...@gmail.com <javascript:>> wrote:
>>>
>>>
>>> On Dec 30, 2013 10:10 PM, "Patrik D&apos;haeseleer" <pat...@gmail.com<javascript:>>

[Cathal Garvey]

Allergy wouldn't be the same reaction.

With GFP exposure outside cells, you'd expect no reaction at first, and
if an allergy results, then on later exposure you'd get swelling, etcetera.

With GFP *inside cells*, you'd expect immune to seek out any cell
bearing the GFP antigen, and trigger apotosis in those cells or
*degranulate on their asses*.

So, not just inflammation, but destruction of cells in the area by the
immune system. If enough cells are destroyed at once, you'll end up with
weakened or necrotic tissue and potentially a carbuncle or open sore.

If you're *really* unlucky, the anti-GFP reaction will cause an
autoimmune cross-reaction against a normal cell antigen, and you'll
develop something like Lupus. But I'll grant you that for GFP, that's
pretty unlikely! I doubt anti-GFP antibodies/TCAs resemble
anti-anything-else-in-the-human-body.

[Mike Horwath]

Cathal you really summed up these issues nicely!  I few more points for people interested in this stuff:

• Some proteins are inherently more immunogenic than others.  Sometimes, they just don't have good epitopes for antibodies to target, and/or fragments from them don't fit well into MCHII molecules for presentation to T cells.  GFP however seems to be pretty immunogenic: http://www.ncbi.nlm.nih.gov/pubmed/10455440
• "Immune Privilege" for the brain and eye is the result of active immune suppression plus physical barriers to immune cell entry (lack of lymphatics, blood-brain barrier).  However, this privilege can and does fail...see multiple sclerosis and symphathetic opthalmia, for example.
  
• I agree that engineering infrared vision is way beyond current capabilities :)
  

Mike (immunology grad student)

[Andreas Stuermer]

I guess I gotta dig into immunology! Especially now that the immunology-lecture at my university has been cancelled for financial reasons.

How can the immune sytem know GFP is new?

What if two humans interbreed and one has a mutant protein? Why doesn't the immune system attack that protein, and does attack GFP??

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[Andreas Stuermer]

I mean the immune system of the resulting baby, of course.

[Cathal Garvey (Phone)]

You can thank your spleen! Essentially the immune system creates virtually infinite T-Cells with random ability to bind antigens. Then, during early immune development, they are tested by special cells which present "self" antigens to them. If they bind self-antigens (natural proteins of the baby, if you will), they are disabled.

If I recall correctly, a similar test, but less rigorous, occurs during the development of a T-Cell response to an infection..Mike? :)

What this means is that a baby born with GFP will be fine: their immune system will test and filter out any T-Cells that attack GFP, same as for other self-antigens, so their immune system will not attack GFP bearing cells. But, adding GFP to someone whose immune system matured prior to GFP exposure is riskier.

I recall early experiments years ago into artificial enhancement of T-Cell maturation to assist anti-cancer vaccines: reprogrammed antigen-presenting cells that *really* activate T-Cells that bind the presented antigen. You could in theory do the reverse, and use APCs that induce anergy (Dormancy/Coma) in T-Cells that bind, helping to induce tolerance, but it's unlikely to work- it only takes one T-Cell escaping anergy to give you pseudolupus. :)

[Nathan McCorkle]

basically the immune system undergoes a sort-of imprinting/learning,
where any immune cells that react with cell-presented proteins are
killed. I'm not sure if this happens more during embryo development,
or is constant throughout life, or if it tapers off.

Since the immune system adapts by VDJ recombination (gene
jumping/randomization), killing off reactionary cells during a safe
time (or in a safe place) makes the odds of the immune system
attacking itself very low.

Eventually I guess VDJ recombination could come up with a new matching
motif/sequence, which could be how these autoimmune things happen late
in life.

http://en.wikipedia.org/wiki/V(D)J_recombination
(http://de.wikipedia.org/wiki/V(D)J-Rekombination)

http://en.wikipedia.org/wiki/Negative_selection_(immunology)

Also:
http://en.wikipedia.org/wiki/T_cell#Negative_selection

On Fri, Jan 3, 2014 at 10:23 AM, Andreas Stuermer

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-Nathan

[Andreas Stuermer]

Ok, so you would have to transfect your spleen too to present GFP to the T-cells?

Summa sumarum, state-of-the-art (diy) gene therapy is likely limited to miRNA/silencing RNAs, to silence oncogenes/virusses?

Because mRNA can't provoke immune reactions?

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[Mike Horwath]

Yep, Nathan and Cathal and have described the basics...

• New T-cells (and B-cells) with randomly generated receptors are produced throughout life, although it does slow down as you get older.
  
• T-cells mature in the thymus and auto-reactive cells are eliminated there...not the spleen :)   ...B-cells mature in the bone marrow, and auto-reactive B-cell also get eliminated.  This is called Central Tolerance.  This is why if you were born with GFP, you would probably not develop an immune reaction to it.  However T-cells are long-lived so if you introduce it later, it's too late for central tolerance.
  
• Central tolerance is not 100% effective...some self-reactive cells get through, especially cells that react to proteins not normally found in the thymus/bone marrow.  This is why we also have "peripheral tolerance".  If the immune system finds an unknown protein, it is actually fairly likely to produce a T-regulatory response that suppresses reactive cells.   However, if there is inflammation going on (as in infection) or it's large dose or especially immunologic protein, T-effector responses will dominate.  The cell interactions that produce either peripheral tolerance or activation happen mostly in the 2ndry lymphoid organs, such as spleen and lymph nodes.
• Peripheral tolerance breaking down is an important part of autoimmune disease.
  
• Immunology sometimes feels more like sociology than biology--lots of cells making chemical "arguments" back and forth, and it can be hard to predict with ones will win and give you tolerance or rejection!
• Cathal mentioned activating T-cells to fight cancer.  This is a great topic and makes me geek out a bit :)  There has been slow progress over the last decade, with some promising clinical trials.  It's a great poster-child for gene therapy.
• Some links...
• Central tolerance:  http://en.wikipedia.org/wiki/Central_tolerance
  
• Peripheral tolerance:  http://en.wikipedia.org/wiki/Peripheral_tolerance
• Example of T-cell therapy for lymphoma: http://www.chop.edu/service/oncology/pediatric-cancer-research/t-cell-therapy.html
• Obligatory XKCD: http://www.xkcd.com/938/
  

Mike

[Mike Horwath]

Saw Mega's post after I made my post.   Basically, it's too late for central tolerance because you already have T-cells likely to react with GFP...transfecting thymus/spleen won't help.  You can try to create peripheral tolerance, say by injecting immunosuppressive cytokines, but even the professionals don't have a good handle on it.  You're pretty much playing russian roulette.

Gene therapy DOES have potential applications beyond miRNA.  Increase expression by adding extra copies of a gene, cut it out entirely with CRISPRs, or put in a new variant of a protein that's similar enough to existing proteins not to create a response.

However, viral self-engineering is really not where DIYbio should be focusing...it's difficult and dangerous, questionably legal, possibly unethical.  I think DIYers can contribute by taking part in the public debate, and helping to develop tools for self-diagnosing whether gene therapy could help a person.  But if you actually want to do gene therapy with viruses...think about entering academia or biotech!

[Nathan McCorkle]

Mike, could you instead of transfecting the thymus/spleen, try instead
for bone marrow? I know bone marrow transplant is effective at curing
HIV, but also that it's quite risky surgery. Maybe a transfection
would be like laproscopy, where they only make a small incision to do
surgery, limiting risk and damage?

Also, even though this stuff isn't DIY-level due to safety concerns,
there are lots of young people here so I do think this discussion is
healthy. Lot's of DIY folk work or sometimes communicate with
academia, so there is plenty of interdisciplinary idea formation
potential here.

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[Alex]

How is it that someone can live without a spleen? Can the body just not adapt to cell changes at that point?

Alex

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[zarpaulus]

Life without a spleen is possible because it's functions aren't absolutely necessary to survival, but without one you are highly prone to infections.

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[Andreas Stuermer]

You mentioned the immune system not attacking nerve cells because the damage would likely be more severe than just having a virus inside.

That's why HSV-1 chooses to "stand-by" in the nerve cells, right?

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[Cathal Garvey (Phone)]

That and because nerves live exceptionally long, so the HSV episomal chromosome (which is all that remains of dormant virus) doesn't need to worry about segregational stability.

Having only nuclear DNA means dormant viruses don't trigger immune response in any tissue: no proteins means no response. For example, dormant HIV is integrated into TCell genomes, and they aren't "visible" until they start producing proteins.

[Mike Horwath]

A few responses here:

Nathan, I absolutely agree this discussion is valuable even if people won't be doing it at home! Guess I just wanted to cover my ass a little and not seem like I was recommending DIY gene therapy :)

Transfecting bone marrow is an interesting idea, depending on what you are trying to do.  If you want to create central tolerance to a new protein like GFP, you could theoretically transfect it into the bone marrow (where B cells mature) and thymus (where T-cells mature).  Then, newly produced T cells and B-cells would be eliminated if they react to GFP.  However, you still have the issue of EXISTING B-cells and T-cells hanging out all over the body.  These can be long-lived, especially "memory" type cells, and there's likely to be a few that will recognize GFP and start multiplying like crazy. 

Transfecting bone marrow for HIV treatment on the other hand sounds exciting to me.  I'm not an expert in this area but...basically curing HIV with bone marrow transplant works by replacing the patient's bone marrow with donor marrow that carries the rare CCR5 delta32 mutation, making them resistant to HIV.  However, this transplant has high mortality risk because it requires irradiating the patient to kill off their current marrow stem cells and immune cells, and then lifetime immunosuprresive drugs to prevent graft-vs-host (GVH) disease, which sometimes still happens anyway. 

Instead, I think you should be able to pull out some of the patient's own marrow cells, transfect them to replace the CCR5 with the mutated version, irradiate the patient, then put the "improved" cells back in.  This would avoid the risk of GVH disease.  If you skipped irradiation, or just injected your viral vector directly into bone marrow, you would end up with an unpredictable mix of original and improved cells.  This could improve immune function but would probably leave some viral load.

RE living without a spleen....Cathal is right that it will increase susceptibility to infection, but it's not actually as bad as you might think.  Remember T cells are produced in the thymus, not the spleen.   Spleen is a major site (in addition to lymph nodes) where phagocytic cells, T cells, and B cells congregate to create responses to infections.  Spleen is especially important for antibody response to blood-born bacterial infections, so you will be more likely to get those infections without one.  The spleen also helps to clear out old/damaged red blood cells, but that function is also not essential.

Wow, that turned into a long post!

Mike

[Katie Kearns]

On Sat, Jan 4, 2014 at 6:28 AM, Andreas Stuermer <masters...@gmail.com> wrote:

You mentioned the immune system not attacking nerve cells because the damage would likely be more severe than just having a virus inside.

That's why HSV-1 chooses to "stand-by" in the nerve cells, right?

Just as an aside, people sometimes *do* react to themselves. That's what auto-immune disorders are. 

Also, pregnant women can react to their babies -- that's what's assumed to be behind some miscarriages. It also causes "rh disease", where the mom's immune system reacts to the rh factor of the baby's red blood cells (if the mom is rh- and the baby is rh+) -- it's treated by giving these moms a shot of anti-RhD IgG around the end of the second trimester. (More reading about immune response in pregnancy: http://en.wikipedia.org/wiki/Immune_tolerance_in_pregnancy )

-Katie

[Andreas Stuermer]

>Then, newly produced T cells and B-cells would be eliminated if they react to GFP.  However, you still have the issue of >EXISTING B-cells and T-cells hanging out all over the body.

So you would have to do two engineering steps. The first is to bring GFP into bone marrow and thymus.

Ten years later, you transfect your skin with GFP and hope that no T-cells with anti-GFP are remaining, right?

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[Mike Horwath]

Hi Mega,
That might work.  I can think of 2 potential issues:

1)  The immune system might react against the GFP in the thymus and bone marrow, eliminating the transfected cells.  You would probably need immuno-suprressive drugs, at least at first.
2)  Even at 10 years there's no guarantee all the reactive cells will be gone.  They might dwindle to zero, but memory cells can last a lifetime.  Especially if there's some GFP-cross-reactive antigen you are exposed to that tickles the GFP-reactive cells periodically causing them to replicate.

IMO it's very hard to tell if it would work unless you tried it...

Mike

[Cathal Garvey]

See Mike's corrections on my rusty Immunology. :)

>>>> - Some proteins are inherently more immunogenic than others.

>>>> Sometimes, they just don't have good epitopes for antibodies to target,
>>>> and/or fragments from them don't fit well into MCHII molecules for
>>>> presentation to T cells. GFP however seems to be pretty immunogenic:
>>>> http://www.ncbi.nlm.nih.gov/pubmed/10455440

>>>> - "Immune Privilege" for the brain and eye is the result of active

>>>> immune suppression plus physical barriers to immune cell entry (lack of
>>>> lymphatics, blood-brain barrier). However, this privilege can and does
>>>> fail...see multiple sclerosis and symphathetic opthalmia, for example.

>>>> - I agree that engineering infrared vision is way beyond current