**2. Rationale for gene therapy in LSDs**

150 Non-Viral Gene Therapy

efficient to some of these diseases, especially if performed early enough to prevent irreversible lesions. Nevertheless, limitations such as the need of an early diagnosis, the difficulties to find a compatible donor in short time, and the high rates of morbidity and mortality associated with the procedure still limit this type of treatment (Malatack et al, 2003). Therefore, despite the many advances in this treatment over the last 30 years (Boelens et al 2010), its use has been deferred in favor of ERT whenever it is available. Enzyme replacement therapy is approved for a growing number of LSD, especially those without CNS involvement. It has proven to reduce some visceral symptoms as hepatosplenomegaly and improve respiratory function (Sifuentes et al, 2007), however difficult-to-reach organs such as the brain and the bones are still a major challenge. Innovative routes of enzyme delivery have been tested to achieve the CNS, such as

Other treatment approaches already under clinical use or experimentation are Specific Substrate Inhibition (SSI) and Pharmacological Chaperone Therapy (PCT). SSI aims to decrease the storage by reducing substrate synthesis through an inhibitor. PCT uses small molecules able to stabilize the mutant enzyme and help it escape proteasomal degradation, thus restoring some residual enzyme activity. All such treatments have limitations (table 2),

Approach Brief Description Pros Limitations

Ameliorates several visceral symptoms

Reaches the CNS

Able to correct visceral and CNS symptoms if performed early

Orally administered Reaches the CNS

Orally administered Reaches the CNS

Orally administered Reaches the CNS Low cost

Potentially effective with single injection

High cost Does not correct difficult-to-reach sites Need of repeated injections

High Cost Need of repeated injections Efficacy uncertain

Limited efficacy Relatively high morbidity and mortality rates

Limited efficacy High cost

Works only in patients with specific mutations

Works only in stopcodon mutations Clinical trials yet to be performed

Safety concerns Efficacy uncertain

intrathecal ERT (Munoz-Rojas et al, 2008; Munoz-Rojas et al, 2010).

that justify the development of gene therapy approaches for these diseases.

Intravenous injection of a recombinant version of the missing enzyme

Intrathecal injection of a recombinant version of the missing enzyme

Non-autologous transplantation of CD 34+ cells

Use of drugs that can inhibit the synthesis of the undegraded material

Drugs that can stabilize the mutated protein, allowing some enzyme activity

Use of molecules that can suppress stop-codon mutations

> Administration of a normal copy of the mutated gene

Table 2. Pros and limitations of different therapeutic approaches for lysosomal storage

Intravenous enzyme replacement therapy

Intrathecal enzyme replacememnt therapy

Hematopoietic stem cell tranplantation

Specific substrate inhibition

Pharmacological chaperone therapy

Stop-codon readthrough therapy

Gene Therapy

diseases.

Abbreviations: CNS – Central Nervous System

The rationale for gene therapy and other enzyme-based approaches for treatment of LSDs was first introduced almost five decades ago by Christian de Duve, and can be summarized in the following sentence from his original work "In our pathogenic speculations and in our therapeutic attempts, it may be well to keep in mind that any substance which is taken up intracellularly in an endocytic process is likely to end up within lysosomes." (de Duve, 1964). His work and other pioneer studies showing cross-correction between fibroblasts from patients with Mucopolysaccharidosis type I and type II (deficient in alpha-Liduronidase and iduronate-sulphatase, respectively) established that the enzyme produced in one cell could be uptaken by a deficient cell, thus restoring its phenotype (Fratantoni et al, 1968). Later studies identified that this uptake was a saturable, receptor-mediated process, and the main actor of this process was the mannose-6-phosphate (M6P) receptor localized in the plasmatic membrane. The post-translational modification of addition of the M6P to the protein was discovered to be a signal not only to endocytosis but also for targeting nascent hydrolases to lysosomes (Fisher et al, 1980; Sly et al., 1981).

These pivotal discoveries in the field of endocytosis and targeting of lysosomal enzymes provided the basis for treatments like HSCT and ERT. In the same way, LSDs may be considered good targets for gene therapy, despite their multisystemic involvement. The correction of a few cells could lead to the enzyme being secreted into the circulation and uptaken by the deficient cells, resulting in widespread correction of the biochemical defect (figure 1).

Since long term gene expression is desirable, most clinical and preclinical trials used viral based vectors. Initial studies on fibroblasts showed promising data using retroviruses (Anson et al, 1992). However, when tests in animal models started, it became clear that some organs as the brain would not be easily corrected, as the enzyme could not cross the bloodbrain-barrier (Elinwood et al, 2004). This is a major hurdle as most LSD shows some degree of CNS involvement. Nevertheless, CNS targeted approaches could be envisaged to overcome this obstacle. For instance, Worgall et al. (2008) showed a slowing of progression of Neuronal Ceroid Lipofuscinosis 2 in ten children treated with serotype 2 adenoassociated virus expressing *CLN2* cDNA. Another clinical trial, for Pompe disease, also used adeno-associated-based vector, but in this case serotype 1 (NCT00976352 www.clinicaltrials.gov). Other two trials performed in the late 1990s used retroviral vectors for Gaucher (Dunbar et al., 1998) and Mucopolysaccharidosis type II (NCT00004454 www.clinicaltrials.gov).

Safety issues related to immune response of the adenoviral vectors (Wilson, 2009) and the possibility of insertional mutagenesis of the retroviral vectors (Hacein-Bey-Abina S et al., 2008) led researchers to develop a series of studies in parallel using non-viral approaches to treat lysosomal storage disorders.
