Although genetically modified mice have provided important new information about the function of many genes, there are serious limitations to current animal models for a number of neurogenetic diseases.
One reason for this is that a mouse ortholog to a human gene of interest may not exist.
It is estimated that between 0.5 – 1% of human genes do not have mouse orthologs.
For example, no mouse ortholog has been identified for the KAL1 gene.
Loss-of-function mutations in this gene cause Kallmann's syndrome, a neurodevelopmental disorder that results in anosmia and hypothalamic hypogonadism.
While mouse orthologs to genes involved in human diseases can usually be found, targeted mutations of these genes in mice may not result in any of the symptoms observed in humans with loss-of-function mutations in these genes.
For example, the HPRT1 gene is mutated in Lesch-Nyhan's disease.
Symptoms include mental retardation, self-mutilation, choreoathetosis and spasticity.
HPRT1 mutant mice do not exhibit the Lesch-Nyhan's disease phenotype.
The most common problem with gene-targeted mice is that they sometimes provide incomplete models of the human phenotype.
For example, children with Ataxia-Telangiectasia (due to mutations in the ATM gene) exhibit ataxia (due to neurodegeneration of the Purkinje cells of the cerebellum), increased incidence of cancer and immune system dysfunction.
Mice with mutations in the ATM gene exhibit increased incidence of cancer and immune system dysfunction, but not degeneration of the Purkinje neurons and ataxia.
Mouse models of Alzheimer's disease also exhibit some but not all of the symptoms of this disease.
Rhesus macaque models of neurogenetic diseases are particularly desirable because the organization of the rhesus brain more closely resembles the human brain than does any non-primate brain.
Monkey brains greatly exceed the size and complexity observed in mouse brains.
Consequently, rhesus macaques exhibit perceptual, cognitive and behavioral plasticity not observed in mice.
Neurological disorders may require novel therapeutic methods, such as gene therapy, which contain significant risks.
The availability of non-human primate (NHP) models will be essential to guarantee safety and efficacy of these new treatment options.
A new approach to genetic modification in mammals
The key to successful gene targeting in mice has been the availability of embryonic stem (ES) cells.
These cells have two important characteristics which facilitate genetic modification:
1. They are immortal and can therefore be propagated indefinitely in cell culture, and
2. They are totipotent and can therefore be used to create chimeric animals.
Some of these chimeric animals will contain germ cells derived from genetically modified ES cells.
Breeding of chimeric animals can thus result in offspring in which every cell is genetically modified.
However, even if such cells were available, the requirement that chimeric animals be bred to produce animals with the genetic modification in all cells would mean that it would take at least 5 years to produce a heterozygote (since macaques do not breed until they are 4-5 years old).
Breeding to produce homozygotes would require at least 10 years.
The basic idea is to perform gene targeting in somatic cells and to then transfer the nuclei of these genetically modified cells to enucleated oocytes.
The resulting embryos are then transferred to a surrogate mother.
The animal that develops from this embryo should contain the genetic modification in every cell of its body.
Application of this approach to rhesus macaques could be used to create NHP models of neurogenetic disease.
Given the difficulty associated with generating a gene-targeted rhesus macaque, it is important to have a set of criteria for disease/gene selection.
The criteria are as follows:
1. Mutation in the gene should cause a serious human disease
2. No complete mouse model should be available
3. The phenotype should be apparent early in the lifespan of rhesus macaque.
Three genes/diseases that meet these criteria are currently exploring: HPRT1/Lesch-Nyhan's disease, KAL1/ Kallmann's syndrome and ATM/ataxia telangiectasia.
Lesch-Nyhan's disease is a neurodevelopmental disorder that was caused by loss-of-function mutations in the HPRT1 gene.
HPRT1 is located on chromosome X.
As a result, most individuals affected by this disease are males.
The HPRT1 gene encodes hypoxanthine guanine phosphoribosyltransferase, an enzyme involved in purine metabolism.
The link between loss of this enzyme and the mental retardation and self-injurious behavior observed in patients with Lesch-Nyhan's disease is currently not understood.
Loss-of-function mutations in KAL1, located on chromosome X, cause some forms of Kallmann's syndrome.
In patients with Kallmann's syndrome, the olfactory nerve is formed but does not enter the telencephalon, resulting in anosmia.
Because gonadotropin releasing hormone (GnRH) neurons originate in the olfactory placode and migrate to the brain early in embryonic development with the olfactory nerve, GnRH neurons form, but do not enter the brain in Kallmann's syndrome patients.
Ataxia-telangiectasia is a neurodegenerative disease that is initially observed in young children.
Loss-of-function mutations in ATM, located on chromosome 11, cause this disease.
ATM is a key player in responding to DNA double-strand breaks.
Patients with ATM are at increased risk of cancer.
Gene targeting in rhesus macaque fibroblasts – problems and solutions
Many neurological disorders are due to loss-of-function mutations.
Examples include Kallmann's syndrome, some forms of mental retardation (e. g. Lesch-Nyhan's disease), ataxia (e. g. Ataxia-Telangiectasia), deafness, blindness and some forms of Parkinson's disease.
Gene targeting will be necessary to create animal models for these diseases.
There are four major barriers to gene targeting in somatic cells: Senescence, lack of isogenic DNA libraries, the impracticality of crosses to get homozygotes, and inefficient gene targeting.
Somatic cells, unlike ES cells, are mortal.
This is a problem for gene targeting as the necessary steps (transfection, selection and screening) require a significant number of population doublings.
Rhesus fibroblasts frequently become senescent during the time in vitro required for gene targeting.
One solution to this problem is the use of human telomerase reverse transcriptase (hTERT).
hTERT reverse transcribes telomerase RNA (TR) to extend telomeres.
The erosion of telomeres during cell division causes senescence in some cell types.
TERT expression is the rate-limiting step in telomere extension.
TERT expression has been shown to immortalize fibroblasts, retinal-pigmented epithelial cells, endothelial cells, keratinocytes, mammary epithelial cells and osteoblasts.
Importantly, transfection of hTERT constructs has been shown to extend the lifespan of rhesus fibroblasts indefinitely.
Further, although there are other methods for immortalizing cells, only TERT immortalizes without transforming cells.
This is important for this approach, as an entire animal will be generated from the nucleus of the cell transferred to the enucleated oocyte.
The unique properties of TERT make it an ideal reagent for extending the lifespan of fibroblasts to permit gene targeting.
However, constitutive TERT expression may alter gene expression in somatic cells.
Because the phenotypic effects of constitutive TERT expression in a primate are unknown, it is important that construct-derived TERT only be expressed in cell culture.
Constitutive TERT expression in sheep embryos created by nuclear transfer did not interfere with blastocyst formation, implantation or early embryonic development but may have interfered with fetal development.
To avoid deleterious effects of constitutive expression of TERT on fetal development, construct-derived TERT should be removed before nuclear transfer.
There is general agreement that the use of isogenic DNA in gene targeting vectors improves targeting efficiency.
For inbred strains of mice, acquiring isogenic genomic DNA is quite easy as genomic libraries from the same strain used to create null mutants is available.
Recently, a rhesus genomic library has been constructed.
However, during the development of null mutant rhesus macaque technology, different cell types derived from different animals will be investigated for efficiency of gene-targeting and nuclear transfer.
It is impractical to create a library for each animal used.
Thus, another method must be used to acquire homologous genomic sequence.
The use of long and accurate PCR provides a solution to this problem.
Homozygotes are required to observe a phenotype for many loss-of-function diseases.
However, rhesus macaques do not breed until they are 4-5 years old.
Thus, producing homozygotes would take a minimum of 5 years using breeding.
One way to avoid this wait is to target X-linked recessive genes in XY cells.
In this case, it will only be necessary to disrupt one allele to see a phenotype.
For autosomal recessive genes, it will still be necessary to disrupt two copies of a gene.
Successive rounds of gene targeting can accomplish this.
The use of TERT-lifespan-extended cells makes this approach practical.
Gene targeting is very inefficient, even in mouse embryonic stem cells.
How efficient is gene targeting in rhesus fibroblasts likely to be?
Since gene targeting has been achieved in fibroblasts for several species, including humans, it is instructive to consider rates of success in these species.
The first report of gene targeting in fibroblasts followed by nuclear transfer occurred in sheep.
A very high level of gene targeting efficiency was observed using Lipofectamine for transfection.
In contrast, low levels of gene targeting efficiency were observed in sheep, pig and human fibroblasts using electroporation for transfection.
The reported high efficiency of gene targeting with Lipofectamine in sheep fibroblasts is surprising given that chemical methods have been shown to be a poor method for gene targeting both in mouse and in human ES cells.
For this reason, electroporation is the gene targeting method of choice for ES cells.
Given an expected low efficiency of gene targeting, it is important to design experiments to maximize chances of success.
For example, it is expected that large numbers of cells will be required.
Fortunately, TERT life span extension means that unlimited numbers of cells are available for experimentation.
The length of homology is another variable, which may influence success in gene targeting.
Not all gene-targeting events result in functional disruption of the gene of interest.
Given the expense and time involved in creating rhesus macaques through nuclear transfer, it is desirable to be as certain as possible that functional disruption of a targeted gene has been achieved prior to nuclear transfer.
One advantage of targeting the HPRT1 gene is that functional selection could be applied.
Other approaches may improve gene targeting in NHPs still further.
For example, although infrequently used, relatively high levels of gene targeting have been achieved with adenovirus vectors.
Gene targeting in somatic cells has been recently shown to be dramatically improved with two experimental protocols:
1. Thymidine block of cell replication and
2. Including a SV40 enhancer sequence in the targeting construct.
Thymidine treatment has been shown to induce homologous recombination.
A 72 bp fragment from the SV40 enhancer improves import of plasmids into cell nuclei.
This sequence apparently allows plasmids to bind to transcription factors, which are transported to the nucleus via nucleus localization signals.
The combination of thymidine treatment and the presence of the SV40 enhancer sequence in the targeting construct allowed to obtain a ratio of homologous recombination to random integration of the construct of 1:3 after electroporating bovine fetal fibroblasts.
The production of genetically modified rhesus macaques poses a number of serious challenges.
The first step is to produce genetically modified cells (Table 1).
The more difficult feat of gene targeting is expected to be greatly facilitated by extending the lifespan of somatic cells with hTERT.
Thymidine treatment and improved nuclear import of targeting constructs with SV40 enhancer sequence may also substantially improve gene targeting in rhesus macaque somatic cells.
A number of rhesus ES cell lines have been derived.
Some of these may prove useful in gene targeting experiments.
The problems associated with nuclear transfer from somatic cells are currently being tackled.
Experience with rhesus embryonic and human somatic cells suggests that there is no unsolvable barrier to whole animal cloning in primates.
Solving the various challenges to producing genetically modified macaques is well worth the effort as null mutant NHPs will likely result in breakthrough advances in the understanding of neurogenetic disease and prove invaluable for preclinical trials of new therapies.
Table 1. Procedural steps in producing a genetically modified rhesus macaque
1. Isolate isogenic genomic fragments using long and accurate PCR|
2. Construct targeting construct
3. Transfect TERT life-span extended cells with targeting vector
4. Select and identify targeted clones
5. Remove TERT
6. Nuclear transfer
7. Embryo transfer
8. Analysis of genetically modified NHP