Gene therapy -- a technological challenge, but we're good at meeting technological challenges

A friend told me recently that gene therapy for diseases of the skin was making a lot of progress.  Given the fits-and-starts history of the field of gene therapy, the fact that the US Institute of Medicine has recommended that most research in gene therapy no longer require the special review it has long been subject to (discussed in Nature this week here), as well as recent announcements of progress and hope, I was interested in looking into it.

Gene therapy, or more generally genetic engineering, is the replacement of a disease-causing allele with a working one, or the silencing of a causal allele.  Gene therapy has a long history, and not always a successful one.  It first looked possible in the early 1970's when Friedmann and Roblin proposed in Science that "gene therapy may ameliorate some human genetic diseases in the future", before much at all about the association between genes and disease was understood. 

There has been much progress since the 70's in understanding the essentials of gene function, of course; the genetic underpinnings of many diseases, primarily rare, have been identified and, while gene therapy has seen some tragic failings, progress is being made.  In theory, while promises in the past were too grandiose, medicine is likely to see many more successes in treating single-gene diseases.  After all, when faced with a clear problem, humans are very good at engineering solutions.

The theory is straightforward: find the gene, figure out what's going wrong, and stop or replace it.  Once a faulty gene has been identified, though, the fundamental problem has been delivering the therapeutic DNA into the nucleus of cells in the affected organ, and then into the right site in the genome so that it can either halt coding for the defective protein or begin telling the cell how to make a working copy of the protein.

Gene therapy using an adenovirus vector: Wikipedia

One way to do the delivery is with a viral vector.  Viruses are naturals at delivering their own genes into host cells, so, in theory harnessing that ability for therapeutic purposes is ideal.  Target the right type of cell, and the introduced gene can be expressed directly or integrated into the host-cell's genome where it will be expressed.  However, most genetic diseases would require repeated delivery of the therapeutic DNA, which would mean repeated exposure to the virus.  Our immune systems are very good at identifying and targeting invading viruses; while this is usually a good thing, it's not so good for virus-based gene therapy that needs to be repeated.  And, if the patient was exposed to the virus before its use in therapy, it's less likely to be effective, again because of the immune response.

Gene therapy makes a comeback
Indeed, it was a severe inflammatory response to an adenovirus-delivered therapy that was responsible for the death of Jesse Gelsinger in 1999, a 19-year old subject in a clinical trial.  His death greatly reduced enthusiasm for gene therapy, and rightly so.  But it did eventually give researchers insight into safer and more effective DNA delivery systems, adeno-associated virus (AAV) being one, although the length of DNA that it can carry is limited, and thus so are the diseases it can eventually be used to treat.  Even so, as reported in a paper in ABBS last year ("Phoenix rising: gene therapy makes a comeback", ABBS (2012) 44 (8): 632-640, Lamberis), AAV-based therapy has been successful now for a genetic cause of blindness, LCA, and there were 80 on-going clinical trials testing the efficacy of AAV-based gene therapy in 2012. 

Retrovirus-based vectors are another approach.  An advantage over AAV is that these can incorporate much larger transgenes, but they have been associated with leukemia-like T lymphoproliferative disorder due to insertional mutagenesis, or random insertion of the virus and its engineered load into the target DNA.  Lentivirus-based vectors are another possibility, but they, too, have their downsides. 

Non-viral vectors consisting of "lipids, peptides, carbohydrates or nanoparticles that fuse with the cell membrane and release the therapeutic DNA in the cell cytoplasm" (Lamberis) depend on natural mechanisms of the target cell to take them in and transport them within the cell.  They aren't as efficient as viral systems, but are safer and less likely to trigger immune responses than virus-based vectors.  

A prime candidate for successful genetic engineering is skin disease.  The skin is an eminently accessible organ, so that if the problem of getting the therapeutic agent through the epidermal barrier is soluable, topical treatment of many skin diseases could be envisioned.  Amy Paller and colleagues reported success with this last year in PNAS.  They introduced "spherical nucleic acid nanoparticles conjugates" into the keratinocytes of mice, and in human skin grafted onto a mouse, with the ultimate goal of delivering RNA silencing systems that can inhibit the expression of faulty genes for a variety of keratin-associated skin diseases.

FIRST, the Foundation for Ichthyosis and Related Skin Types, awarded Paller with funding to continue with this work.  The foundation's announcement describes Paller's work this way: 

The blistering and thickening of skin seen in EI [epidermolytic ichthyosis] usually results from a change in a single letter of the DNA code (a mutation) in one copy of the gene that provides the codes for manufacture of a keratin protein in the upper layers of skin. Small interfering RNAs (siRNAs) are small pieces of genetic material that can identify DNA pieces and bind to them, preventing the gene from being translated into protein. siRNAs are able to distinguish the mutated DNA from the normal DNA, and thus are able to prevent only the abnormal keratin protein from being formed. The problem with siRNA has been getting it through the skin barrier to where it needs to go. Dr. Paller and her team have found a way to get the siRNAs through the skin, through nanotechnology. By putting about 30 copies of the siRNA all around a central gold nanoparticle (leading to what her group calls “spherical nucleic acids”), the siRNAs are able to be rubbed into skin in a simple moisturizer.

This indeed sounds as though it has potential.

CRISPR -- the next big thing
We can't end this post without mentioning another technological advance getting a lot of attention these days.  It exploits a portion of some prokaryotic immune systems, CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats.  Found in bacterial and archaeal genomes, these short repeat DNA sequence loci, from invading structures called phage or plasmids, get incorporated into the prokaryote's genome between CRISPR repeats, as a 'search' sequence.  The CRISPR resulting structure is then the basis for recognition of exogenous invading genomes. The structure moves along an incomer's DNA till it detects a match to the incorporated 'search' sequence.  Then, aided by genes called CAS genes, it cuts the detected DNA.  Normally that destroys it, but other enzymes can be targeted to this cut, and repair it.  Here's where genetic engineering comes in -- the repair process can insert a user-designed sequence into the targeted break.

As Elizabeth Pennisi described in her piece, "The CRISPR Craze", (Science 23 August 2013 341(6148), 833-836), CRISPR are also showing potential for use in gene therapy if they can be used to "delete, add, activate or suppress targeted genes in human cells". The idea in general is that a harmful sequence could be detected, the DNA cut at this point, and a 'good' sequence inserted to replace the harmful part. Here's a video that shows the idea.  Mark Wanner at Jackson Labs also nicely describes the potential uses of CRISPR on his blog. 

There is much excitement about CRISPR's potential.  A story in The Independent quoted several scientists the other day, among them George Church, a geneticist at Harvard, who was one of the first to use CRISPR to actually edit nucleotides in human sequence. "“The efficiency and ease of use is completely unprecedented. I’m jumping out of my skin with excitement,” said Church."  Ok.  But there is still a lot of work to be done.  Getting the edited gene to the right place in the target genome without undesirable effects elsewhere will be a challenge, as with conventional gene therapy.

But, if humans are good at anything, it's technology, and gene therapy and genetic engineering are primarily technological challenges.  We often criticize the science of genetics as it portrays itself and its successes, but if these technologies live up to their potential, they can change a lot of lives for the better.