As we begin the sixth year of “The Primer” series, we’re going to take a detour out of molecular diagnostics for this month’s installment and cross into the realm of gene therapy. Once the stuff of science fiction, the ability to permanently correct or compensate for an underlying genetic disease through the introduction of replacement genetic material marks the ultimate pinnacle of personalized medicine, and it has made slow but significant progress over the past few decades.
To appreciate how deliberate that progress has been, consider that the first successful experimental human gene therapy subject, suffering from an immune deficiency known as adenosine deaminase deficiency (ADA), was treated in 1990; but in the United States it wasn’t until 2017 that the first gene therapy (Kymriah) achieved FDA approval. As the tools and methods used to conduct gene therapy become further refined, the number of therapies will slowly increase, and molecular laboratorians will of necessity become involved in aspects such as monitoring treatment success.
As always, the goal of The Primer is to demystify aspects of the underlying technology for the laboratorian. With that in mind, this month we’ll examine one particular approach to gene therapy, both in itself and as a model for how one general class of gene therapy (viral vector methods) works.
Defining the challenges
First, let’s review a few of the bigger challenges in gene therapy. Gene defects can occur through non- or under-expression of a gene, or through expression of a non-functional form of a gene, in a form which is recessive in Mendelian genetics terms; that is, in the presence of a “good” gene copy, the defect is masked or corrected for. Or, they can occur through ectopic (wrong cellular context) or over-expression in a form which is more akin to a Mendelian dominant trait (where presence of a “good” gene copy won’t help).
While that’s rather a gross oversimplification of all the nuances possible within genetic diseases, it’s a useful broad dichotomy and one from which it is probably apparent that some types of mutations (“recessive”) are easier to address than others (“dominant”) by addition of extra “good” gene copies to the patient. Even in such cases, however, if the gene in question should normally only be active in limited cell populations and in response to particular cellular stimuli, additional hurdles have to be surmounted to get the supplemental “good” gene and its regulatory sequences into the appropriate cell type.
An easier scenario is one in which the missing or defective gene product doesn’t require expression regulation—a steady, constitutive expression is suitable—and ideally, where the product is something such as a secreted product, so that its expression by even a subset of cells can provide needed biological activity to all nearby cells. Of course, very few genetic diseases fit all of those criteria for an “easiest case.” Also, we want to have a strong assurance that our insertion of new genetic material is done in a controlled fashion so that it doesn’t randomly insert with potential to disrupt other critical genetic loci. (You may be starting to appreciate why the pace of gene therapy development remains slow…).
Viral vectors
With those factors kept in mind, in all cases gene therapy must hinge on finding a controlled method for getting the desired copy of a supplemental gene and any required regulatory sequences into the host genome, and perhaps even into only a particular desired subset of cells. This gene introduction should be both efficient (that is, occurring in a significant proportion of target cells) and stable (allowing for long-term expression of the supplemental gene, ideally carried forward into any cells resulting from division of the originally modified cells). Luckily, nature has provided tools with exactly these capabilities in the form of several types of viruses—small exogenous carriers of genetic material, many of which are specific as to the type of cell they will introduce their genetic package into and proceed to integrate that package into the infected cell genome. Doing that establishes the introduced material stably, as the cellular DNA replication machinery will now replicate it as an integral sequence in a chromosome, allowing for somatic passage. While a few classes of virus are particularly promising as gene therapy tools (“vectors,” or carriers of supplemental genetic material), we’ll choose just one particular virus type—adeno-associated virus, or AAV—as our explanatory model.
AAV exists as several closely related serotypes, which we’ll consider together, as their main difference is in host cell tropism. It is a member of the Parvoviridae family, and it’s tiny, with a roughly 18 nm diameter, unenveloped icosahedral protein capsid carrying ~4700 bases of DNA. Note that I wrote bases, not base pairs, because other than palindromic hairpins at either end required for the viral replication cycle, AAV is single-stranded and can be either (+) or (-) polarity.
AAV is attractive as a tool for moving engineered pieces of DNA (or at least short ones, less than ~4000 bases) into target cells for several reasons. First, it can infect a wide range of either resting or dividing human cells efficiently, including neurons, skeletal muscle, hepatocytes, and vascular smooth muscle. Upon doing so, it has the ability to insert (integrate) its DNA into the host cell genome after its convenient conversion to a double-stranded form by host cell enzymes. Even better, in the majority of cases, this integration occurs at a known site on chromosome 19 where it doesn’t cause problems. (In fact, AAV is not associated with any known disease in humans, and is only very mildly immunogenic.) Finally, on its own it’s a “replication-defective” virus; it can’t spread further without the presence of another actively dividing helper virus—most commonly, an adenovirus (thus AAV’s name). These attributes combine to make AAV an effective, easily manipulated tool for gene therapy applications whose only major drawback is the limited size of its genetic “payload.” In many cases this size limit is acceptable, however, and AAV-based methods account for a very significant proportion of all gene therapy methods under investigation, with well over 100 clinical trials worldwide having been reported as either ongoing or completed.
The therapeutic approach
While each of these trials would be unique, the general approach to using AAV as a treatment would be similar in all trials. First, as noted earlier, the condition to be treated would probably be a recessive, loss-of-function condition in a single gene with required activity in one of the cell types susceptible to AAV infection. A functional replacement copy of the gene would be cloned into a plasmid, most likely in the context (that is, the relative positioning) of the normal, wild-type associated promotor sequence; that will help to confer correct timing and dose of gene expression once integrated. This plasmid, carrying this intended “transgene,” would be introduced into a tissue culture “packaging cell line” that has been engineered to also express the required AAV viral capsid proteins and the required “helper virus” genes. Nuances on this generic approach include selection of the AAV capsid genes from the AAV strain with the best tropism for the intended target cell type, and differences in the packaging cell line and its method for expression of the viral capsid and helper virus functions, but the end results are the same. Co-expression of all of these together will result in copies of the transgene being created and packaged by AAV capsid into virus-like particles, and the release of these into the cell culture medium where they can be isolated and purified.
With these engineered virus-like particles in hand, the next step is to introduce them into the patient, and ideally, as close to the intended target cell type as possible. That sentence simplifies what in reality may be quite a complex issue on its own; for example, due to the good neuronal tropism of some AAV strains, some neurological conditions, including Parkinson’s disease, have been the subject of AAV-based trials. In that case, microinjection into the relevant brain area is clearly more technically challenging than when the target is a cell type readily reached by peripheral blood circulation, such as cardiac vascular smooth muscle (the target of another AAV-based trial, relating to heart failure). In between these two extremes of accessibility lies a spectrum of delivery challenge.
Regardless of how delivery is achieved, the influx of transgene carrying virus-like particles to the target region results in the “infection” of a significant proportion of host cells. Upon entering the cell, the virus-like particles uncoat, the transgene is converted by cellular polymerases to a dsDNA form, and a portion of these proceed to integration of the transgene into the previously mentioned site on chromosome 19, known as the AAVS1 site. Recall that AAV was able to do this whether this host cell was currently quiescent or replicating, but in either case, should this cell replicate in the future, all of its progeny cells will carry the integrated transgene. Recall also that AAV has generally low immunogenicity, so there was unlikely to be any major immune response to the introduction of the virus-like particles, and that these particles are incompetent of further replication, since they neither have a helper virus nor are carrying the normal AAV genes. Together, these factors have combined to create a safe process in which we don’t risk a scenario of unwanted ongoing transmission of an engineered virus.
So what happens to the transgene, once integrated into its host cells? Recall that we included the native promotor and control sequences with the replacement gene; these are signal-responsive DNA elements, which “sense” the intra- and extracellular environment through an array of pre-existing cell signaling mechanisms. These now respond as the cell would intend, signaling for the production of the gene product as, where, and when needed; the transgene product is produced, and by nature of the treated issue being recessive, this positive expression of correct gene function can replace that of the inborn defective gene. As the modified cells age and replicate to replace themselves within their respective organs, the transgene persists, and thus a single treatment is expected to be effective for the life of the patient.
There are a number of viral vector systems other than AAV, such as adenoviral-based vectors and lentivirus-based systems, each of which has individual strengths and weaknesses with regard to possible transgene size, ease and safety of use, cellular tropism, and other factors. As a general model, however, the factors considered here in determining whether a given genetic condition is a good target for viral gene therapy, and the overall mechanistic path of creation of replication-defective virus-like particles bearing the desired transgene followed by infection of intended cell targets either in vivo or in vitro with autologous cultured cells and their subsequent re-introduction into the patient, are similar. All of these methods make use of the evolved capacity of viruses to deliver genetic material with efficiency and specificity, turning pathogens into tools of medicine to bring the hope of permanent cures for a selection of maladies.
John Brunstein, PhD, is a member of the MLO Editorial Advisory Board. He serves as President and Chief Science Officer for British Columbia-based PathoID, Inc., which provides consulting for development and validation of molecular assays.
