Efficient, Mutation-Free, Large-Payload Gene Therapy of iPS

Efficient, safe methods of gene therapy will be essential enabling technologies for the repair or obviation of several of the cellular and molecular lesions driving age-related disease and dysfunction. A recent paper from the Scripps Institute demonstrates a major step in this direction with the successful use of helper-dependent adenoviral vectors to rescue cells defective in lamin A, without detectable mutational side-effects.

Efficient, safe methods of gene therapy will be essential enabling technologies for the repair or obviation of several of the cellular and molecular lesions driving age-related disease and dysfunction, notably the accumulations of mutations in mitochondrial and nuclear DNA (including the medium-term obviation of the latter through WILT), as well as the introduction of novel lysosomal hydrolases to clear out age-associated intracellular aggregates.

As we’ve discussed in previous entries on the progress of gene therapy, zinc finger nucleases (ZFNs)  are amongst the most promising methods under current investigation for human use. Even ZFNs, however, have some potential limitations. Perhaps the most important such limitation is that they rely on introducing double-strand breaks in the host genome, which are then repaired by exploiting the native Non-Homologous End Joining (NHEJ) DNA-repair machinery to insert an user-supplied DNA repair template for the novel gene. As such, the potential exists for even so high-precision a method as ZFNs to damage or disrupt non-target genes, introducing mutations or chromosomal aberrations. The potential is especially high in genes located at mutational “hotspots,” whose sequence specificity or structural or functional features make them particularly vulnerable to mutation in interaction with the repair and replication machinery of the cell.

These possible problems might ultimately manifest to a degree that proves unacceptable for direct use in somatic gene therapy; even in the case of cells modified ex vivo, where screening could potentially eliminate such abnormalities before therapeutic use, a high frequency of sporadic mutations would lower the net efficiency and power of the technique. Moreover, as with other nonviral approaches experiments to date, the use of ZFNs to modify the genomes of induced pluripotent stem cells (iPS) has thus far been characterized by low efficiency rates in what is already a low-efficiency technology; this is a significant limitation in itself, and might be further worsened if essential modifications of donor cells could only be achieved with methods associated with high rates of sporadic mutation.

In work just released in electronic form,(1) researchers working under Dr. Juan Carlos Izpisúa Belmonte at the Scripps Institute Center for Regenerative Medicine, have provided a strong proof-of-principle for the advantages of gene editing of iPS using helper-dependent adenoviral vector (HDAdVs), an approach that had already been shown to allow for efficient and precise gene editing in human embryonic stem cells, based on homologous recombination (HR).(4) This technology uses so-called “gutted” adenoviral vectors, generated by removing large sequences of viral DNA essential to the replication and packaging of the pathogen genome, and replacing their function with proteins from a helper virus or cell line in trans. This offers the simultaneous advantages of removing toxic or immunogenic viral proteins from the vector, eliminating the risk of mutations from double strand breaks, and opening up space for a large (~37 kB) payload of insert DNA.(2)

To test the efficacy of HDAdVs in introducing transgenes to iPS cells, the Scripps investigators chose as a strong test case a defective copy of the gene LMNA,  which encodes lamin A, one of the proteins that make up the nuclear lamina, and which is thought to be involved in regulating gene expression, the stability of the nucleus, and chromatin structure. Inherited mutations in LMNA are responsible for Hutchinson-Gilford Progeria Syndrome (HGPS, commonly referred to as “progeria”); these mutations produce a truncated splicing defect of the protein, which accumulates and leads to nuclear defects including disorganization of nuclear lamina and loss of heterochromatin, resulting in a range of clinical signs and symptoms and early death in patients. While HGPS has been misleadingly characterized as “premature aging” on the basis a subset of the disease’s phenotypes, there is likely no special need to target the gene as part of a panel of rejuvenation biotechnologies; however, its situation on a known mutational hotspot made it an excellent test target for HDAdV in iPS cells. Happily, Belmonte’s group had recently established an iPS line from patient fibroblasts which harbors the mutant protein.(3)

High Efficiency

After transfection, the Scripps team evaluated the efficiency of integration using an inbuilt negative selection system activated by ganciclovir, screening out cells that had been subject to random integration. To their surprise, they found a high (78-100%)  efficiency of integration using HR, even at very low multiplicities of infection, leading to successful integration of the transgene and correction of the mutation in 48% of HR cells.(3)

Successful Reprogramming Without Introduced Abnormalities

Importantly, the corrected HGPS-derived cells were by every test equivalent to wild-derived iPS. They exhibited a normal karyotype, expressed standard markers of pluripotency, had appropriate demethylation of the OCT4 promoter, and appeared to be pluripotent. Moreover, several tests of the HGPS-derived iPS vs. donor fibroblasts revealed no differences in single nucleotide polymorphisms (SNPs), no evidence of gene duplications or deletions; similarly, there were no detectable differences in gene expression profiles by DNA microarray analysis or of methylation patterns between HDAdV-corrected and -uncorrected HGPS-derived iPS.(3)

Phenotypic Rescue

However, LMNA is transcriptionally silent in iPS, so the correction of the mutant gene by the HDAdV system could only be demonstrated in differentiated cells derived from corrected HGPS-iPS. Accordingly, Belmonte’s group differentiated the corrected HGPS-iPS into fibroblasts and smooth muscle cells (SMCs). These cells did not produce the truncated mutant lamin A protein, and did not exhibit the characteristic senescent phenotype of HGPS cells; this included a substantial reduction in staining with staining for senescence-associated beta-galactosidase (SA-β-gal), which was reduced from 21.3% in SMCs derived from uncorrected HGPS-iPS to 6.8% in SMCs derived from corrected HGPS-iPS (comparable to the 11.4% in observed in wild-type BJ-iPS-derived cells), as well as a >60% reduction in the number of abnormal nuclei.(3)

Use in Adult Stem Cells

Buoyed by their success in correcting patient-derived iPS, the investigators tested the HDAdV vector in mesenchymal stem cells (MSCs), selected because the LMNA mutations responsible for HGPS most prominently affect mesoderm-derived tissues such as muscle and adipocytes, and because of MSCs’ widespread use in early-stage regenerative medicine applications. Olfactory ectomesenchymal stem cells were chosen in particular to take advantage of their high proliferation rate, facilitating rapid clonal expansion. Wild-type cells were used due to the inavailability of HGPS-derived MSC. As with HGPS-iPS cells, the HDAdV system achieved high (54%) gene-editing efficiency in wild-type olfactory ectomesenchymal SC, and with no disruption of the native lamin A/C.(3)

A Contender for Use in Human Rejuvenation

HDAdV offers promise for the correction of genetic defects, and for the introduction of novel genes into cells for cell therapy and engineering new tissues impervious to the accumulation of a range of age-related cellular and molecular lesions. There is no reason to expect that a single system will be well-suited to all of the different gene therapy applications that will eventually form part of a comprehensive human rejuvenation strategy. The new system has specific strengths where high-efficiency introduction of large transgenes is required, or where loci of interest are especially vulnerable to disruption by other transgene vectors. Thus, either ironically or appropriately depending on one’s point of view, this proof-of-principle in cells derived from a genetic defect commonly mistaken for an acceleration of the “normal” aging phenotype, may yet ultimately be harnessed to free aging persons from the dysfunction, disease and death that follow from the degenerative aging process.


1: Liu GH, Suzuki K, Qu J, Sancho-Martinez I, Yi F, Li M, Kumar S, Nivet E, Kim J, Soligalla RD, Dubova I, Goebl A, Plongthongkum N, Fung HL, Zhang K, Loring JF, Laurent LC, Izpisua Belmonte JC. Targeted Gene Correction of Laminopathy-Associated LMNA Mutations in Patient-Specific iPSCs. Cell Stem Cell. 2011 May 18. [Epub ahead of print] PubMed PMID: 21596650.

2: Mitani K, Graham FL, Caskey CT, Kochanek S. Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3854-8. PubMed PMID: 7731995; PubMed Central PMCID: PMC42060.

3: Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J 3rd, Izpisua Belmonte JC. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature. 2011 Apr 14;472(7342):221-5. Epub 2011 Feb 23. PubMed PMID: 21346760; PubMed Central PMCID: PMC3088088.

4: Suzuki K, Mitsui K, Aizawa E, Hasegawa K, Kawase E, Yamagishi T, Shimizu Y, Suemori H, Nakatsuji N, Mitani K. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors. Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13781-6. Epub 2008 Sep 3. PubMed PMID: 18768795; PubMed Central PMCID: PMC2544531.

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