In parallel with the development of safer viral vectors, scientists have focused on alternative approaches to achieve disease correction by gene therapy using strategies that would avoid deregulated expression of other genes. True “in situ” gene correction and targeting of safe harbors are receiving particular interest.
Gene correction relies upon replacing the region of the gene containing the mutation with a fragment of DNA containing the wild-type sequencing, while leaving all neighboring sequences intact. If successful, this strategy will “repair” the mutation and allow for normal protein expression. This strategy also has the inherent advantage of using the endogenous promoter and enhancer regulatory elements, thus maintaining physiological expression of the gene. This approach would be particularly attractive for correction of PIDs due to mutations of tightly regulated genes (such as CD40 ligand, CD40LG
), and would also be of special interest to correct disorders that have proven refractory to conventional approaches of gene therapy. One group of such disorders is RAG1 deficiency (associated with SCID, Omenn syndrome and various forms of leaky SCID in humans). Attempts to cure Rag1−/−
mice by RV-mediated gene transfer have failed, unless stem cells are transduced at high multiplicity of infection to integrate a high copy number of the transgene, but this approach exposes to the risk of insertional mutagenesis and lymphoma development11
. Furthermore, even the addition of known endogenous Rag1
regulatory elements in the RV construct does not permit correction of the leaky SCID phenotype of Rag1S723C/S723C
mice (Notarangelo LD and Mostoslavsky G, unpublished observations). Finally, “in situ” gene repair should be considered for rare forms of PID resulting from dominant-negative mutations.
An alternative approach to gene therapy, that would minimize risks of insertional mutagenesis, is based on targeting of specific and safe loci in the genome (“safe harbors”), that are devoid of oncogenes and whose disruption does not lead to deleterious consequences. This approach may be particularly interesting for PIDs, because in most cases PID-causing mutations are scattered through the diseased gene, making gene repair by use of specific HEs or ZFNs unpractical. Potential candidates for locus-specific “safe harbor” targeting include the human Rosa26 locus on chromosome 3 and the Adeno-Associated Virus integration Site 1 (AAVS1) on chromosome 19q13, that encodes for the ubiquitously expressed PPP1R12C gene.
Both gene repair and specific targeting into “safe harbors” require insertion of exogenous DNA sequences into the host genome. Most of the strategies that are used to achieve this goal utilize the endogenous DNA repair mechanisms that are based on homologous recombination (HR). This is one of the critical mechanisms responsible for repairing DNA double-stranded breaks (DSB) that result from exposure to alkylating agents, radiation, but that can also occur spontaneously, especially during chromosome replication. By harnessing the second copy of the affected gene that is coded on the sister chromatid as a template; the HR mechanism facilitates the replacement of the DNA surrounding the DSB by recombination (). If extra-sequences flanked by 5′ and 3′ homology arms are included in the template, HR may promote insertion of these sequences into the repaired site. This mechanism has been historically used for the creation of knock-out and knock-in animal models. Similarly, spontaneous HR may induce gene correction when a template carrying the normal sequence, but otherwise homologous to the targeted mutated area, is introduced into the cell. However the rate of spontaneous HR is extremely low (< 1/106 cells), and is even lower in HSCs than in embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). To circumvent this problem, novel strategies have been designed to introduce locus-specific DNA breaks and promote introduction of exogenous DNA templates into the genome.
Novel mechanisms for gene correction and insertion
Homing endonucleases (HEs) or Meganucleases are highly efficient, sequence-specific enzymes that induce DSB in specific loci. Originally identified in yeasts almost 25 years ago, HEs may function in various cell types derived from different organisms including plants, bacteria, yeasts, Drosophila
, mice and humans, and promote HR by inducing DSBs, with a frequency that is more than 1000-fold higher than spontaneous HR12
HE-specific DNA target sites may range from 14 to 40 base pairs. Given the length of the recognition sequence, only few target sites are expected in the genome; in fact there is only one recognition site for I-SceI (the prototype of HEs) in the whole yeast genome. The LAGLIDADG family of HEs has been studied in greater detail. Its naturally occurring members are either monomeric enzymes with 2 subdomains that target nonpolyndromic DNA sequences or homodymeric proteins that target palindromic sequences. By mutagenizing in vitro the residues of I-CreI HE that are known to mediate recognition and interaction with DNA target sequences, and using high-throughput combinatorial screening, it is possible to generate and characterize a large series of engineered HEs with distinct target specificity. Using this approach, a series of HEs that specifically recognize the human RAG1
locus immediately upstream of the single coding exon 2 have been generated, and their ability to induce DNA repair has been demonstrated upon co-transfection of human cells with plasmids carrying the RAG1
-specific recognition site and a repair matrix13
. The efficiency of targeting is estimated to range from 0.1 to about 5% in human cells (unpublished data). While these data are encouraging, further studies are needed to determine the efficiency of targeting and the ability to induce correction of the endogenous RAG1
locus. Furthermore since the target sequence of the RAG1
-specific HE is not conserved among species and is unique to humans, appropriate “humanized” animal models in which the mouse Rag1
locus is replaced by the human gene (in its wild-type or mutated sequence) must be developed before clinical trials can be proposed.
Zinc-finger nucleases (ZFNs) are a group of artificial fusion proteins that are generated by linking a zinc-finger DNA-binding domain that recognizes a specific DNA target to the nuclease domain of the endonuclease Fok1. ZFNs can be engineered to mediate specific targeting of a mutated gene. The introduction into the cell of a DNA wild-type template may induce gene repair by HR or through non homologous end-joining (NHEJ) (), another cellular mechanism of DSB repair that however is error-prone. Using this approach, correction of a mutation in the IL2RG
gene (mutated in X-linked SCID) was demonstrated in vitro in 5-17% of patient-derived cells14
. Furthermore, ZFNs have been successfully used to mediate targeting of other disease loci in both human ES cells and iPSCs, thus opening the way for future studies and possible applications in human gene therapy15
. However, for both HEs and ZFNs further studies are needed to investigate in greater detail the risks of undesired toxicity due to off-target cleavage. These risks include cell death and the formation of break-induced DNA sequence alterations due to NHEJ as well as chromosomal translocations possibly leading to oncogenic transformation. Finally, cellular delivery of HEs and ZFNs must be also considered. Use of RV or integrating LV vectors may re-introduce the risk of insertional mutagenesis; furthermore, prolonged expression of the HE or ZFN genes may cause deleterious effects as mentioned above. For this reason, NILV vectors that allow expression of the enzyme only for limited time appear attractive, although this type of vector allows less efficient expression of the gene than integrating vectors.
Transposons and transposases
Transposons are DNA elements that are able to spontaneously translocate from a specific chromosomal location to another16
. They can either move directly as DNA or undergo transposition via an RNA intermediate (retrotransposons). This unique activity is facilitated by the transposase protein that targets any DNA cargo sequence flanked by the inverted terminal repeat (ITR) sequences (the transposon). As a result of this process, a DNA fragment is excised from the donor locus and is re-integrated in into another locus (), making this system a good candidate to use as a possible gene delivery system. In order to transform this naturally occurring system into a novel gene delivery tool, a matrix that allows for the expression of the transposase as well as a donor vector containing the gene of interest (flanked by ITR sequences) need to be introduced (separately or jointly) into the host cell in order to achieve gene insertion.
Among transposon systems that are active in human cells and hence may have therapeutic relevance, the Sleeping Beauty
) transposon seems particularly promising, since it exhibits a random pattern of integration that is not biased toward integration into gene loci; furthermore, it is not associated with recombination or deletion events at the integration sites17
system is considered less efficient then the SB; however, it has several advantages: a) it is able to transfer genes of up to 11 kb with minimal loss of transposition activity; b) it creates single-copy insertions; and, c) it is known not to cause gross rearrangements around the integration sites (reviewed in 18
is another transposon system that was recently shown to catalyze transposition in human and mouse somatic cells with higher transposition efficiency then SB
in different cell lines. However, its genomic integration profile is similar to what observed for integrating viral vectors (reviewed in 18
While the transposon technology has already entered clinical trials to induce expression of CD19 chimeric antigen receptors in cytotoxic T cells from patients with refractory B lymphoid malignancies, significant limitations remain for a possible use of this technology in gene therapy of PIDs. In particular, delivery of transposon and transposase constructs into somatic cells still depends on plasmids and/or viral vectors. Moreover, further studies are needed to gain better control of integration sites and to avoid intragenic insertion.
Limitations and Disadvantages
As for any novel technology, several limitations still exist that need to be addressed prior to clinical implementation of the novel approaches to gene therapy discussed here (). Many of these imperfections have to do with specificity and efficiency. In particular, ZFN- and HE-specific recognition sites are not continuously distributed along the genome, thereby limiting the number of mutations that can be targeted and corrected. Furthermore, the introduction of DSBs mediated by ZFNs and HEs may result in a significant risk for chromosomal translocations; this risk is even higher if the enzyme shows poor specificity leading to off-site targeting. Of note, the frequency and nature of translocations (“translocatome”)19
caused by ZFNs and HEs will have to be assessed individually for each of these enzymes used to introduce DSBs. Finally, significant improvement is needed to increase the efficiency of intranuclear delivery of the desired machinery (ZFNs, HEs, etc) and the repair matrix, HE, ZFN etc), while limiting the risk of cellular transformation or dysregulation of gene expression. This goal might be achieved using NILVs to deliver HEs and ZFNs, however this approach currently suffers from low levels of expression mediated by NILVs.
Advantages and limitations of novel approaches to gene therapy
Perhaps the major limitation in the translation of these approaches from the bench to the bedside consists in the lack of appropriate animal models. Therapeutic ZFNs and HEs are designed to recognize and cleave human DNA sequences. Hence, both the efficiency and the safety profile of these approaches can be hardly studied in animal models. A possible strategy to overcome this impediment is the use of “humanized” animal models in which the mutated human gene of interest is introduced to replace the mouse orthologue gene. Despite these limitations, there is increasing interest in the development of novel strategies to gene therapy based on gene correction or targeting of safe harbors.