Promising therapeutic approaches for LSDs are in development. However, there are only isolated instances where a single approach corrects most of the biochemical, histological and clinical features of the disease. This is likely owing to the complex nature of these diseases, as well as the inaccessibility of certain tissues, primarily the CNS. In addition, most of the promising results have been observed under carefully controlled laboratory conditions. Although the primary insult in LSDs is a single-gene defect, multiple secondary mechanisms play a role in the pathogenesis (72
). These include accumulation of secondary metabolites, altered calcium homeostasis, oxidative stress, inflammation, abnormal lipid trafficking, increased autophagy, ER stress, unfolded protein response and autoimmunity. Each of these secondary effects is a potential therapeutic target (Fig. ). It is likely that one or more of these pathogenic mechanisms are at play by the time therapy is initiated, limiting the efficacy of the primary approach. Therefore, several groups have begun combining therapies in order to target either different aspects of disease or different tissues. Additionally, the timing of various therapies can, and has been optimized in order to take advantage of the strengths of each approach. Interestingly, both additive and synergistic effects have been documented when two or more treatments are combined (Table ).
Figure 1. Pathogenic mechanisms involved in LSDs. The disease mechanisms can be divided into primary (red) and secondary (blue) defects. A few or nearly all of these mechanisms may be present, depending on the specific LSD. Each defect represents a possible therapeutic (more ...)
Pre-clinical combination therapies for LSDs
In one of the first examples of combination therapy, weekly ERT beginning at birth was followed by bone marrow transplant (BMT) at 5 weeks of age in the murine model of MPS-VII (73
). The goal was to provide an immediate source of enzyme through ERT while delaying BMT until the deleterious effects of harsh conditioning during the newborn period could be minimized. Although each therapy alone was relatively effective when initiated early in life, the addition of BMT to ERT resulted in a more widespread enzyme distribution and reduced lysosomal storage in the bone, meninges, cornea and retina. In addition, the radiation-induced damage observed when BMT is performed in the neonatal period was avoided.
Substrate reduction therapy (SRT) has been utilized in several LSDs. The goal of SRT is to reduce the accumulation of metabolites by inhibiting their synthesis. As the synthesis of substrates cannot generally be shut down completely, this approach alone will likely only slow the inevitable progression of disease. In the murine model of Sandhoff disease (β-hexosaminidase A deficiency), SRT can be achieved with N
-butyldeoxynojirimycin (miglustat), which inhibits the first committed step in glycosphingolipid synthesis (74
). This effectively reduces the accumulation of total brain ganglioside and GM2 in the mouse (74
). Combining SRT with BMT resulted in a synergistic improvement in lifespan (76
). In the same model, SRT was combined with anti-inflammatory therapy (indomethacin, aspirin or ibuprofen) and antioxidants (α-tocopherol acetate and l
-ascorbic acid). These combinations resulted in an improvement in the lifespan (77
), with synergy demonstrated in animals treated with aspirin and SRT. Injection of neural stem cells into the CNS also synergizes with SRT in Sandhoff disease (78
). In this case, the neural stem cells act as a persistent source of enzyme, cross-correcting the surrounding cells (Table ).
In the mouse model of Niemann-Pick A disease, CNS-directed AAV2-mediated GT combined with an i.v. injection of AAV2/8 expressing human acid sphingomyelinase (ASM) resulted in a synergistic improvement (79
). Through this bimodal approach, the group treated the CNS and systemic disease separately, but simultaneously. At 54 weeks of age, survival in the combination treatment group was 100%, while none of the animals that received either CNS-directed AAV2 or systemic AAV2/8 alone survived. Interestingly, in the combination group, no antibodies against human ASM were detected. The AAV2/8 vector utilized a liver-specific promoter which, combined with the strong tropism of AAV2/8 for the liver, reduced the immune response to the enzyme (80
). Through this approach, the combination of widespread systemic and CNS expression of ASM, as well as the reduced immune response likely explains the synergy observed in this study.
In the profoundly demyelinating and neurodegenerative disease, globoid-cell leukodystrophy (GLD), several combination therapies have been attempted in the murine model (the twitcher mouse). When BMT is combined with SRT (l
-cycloserine), there was an improvement in the mean lifespan to 112 days from ~50–56 days using either therapy alone (81
). Similar results were obtained when CNS-directed AAV2/5-mediated GT was combined with neonatal BMT. There was a dramatic synergy compared to either therapy alone (56
), increasing the median lifespan from 45–55 days to 105 days. In a follow-up study, the median lifespan was further increased to ~130 days when intrathecal administration of AAV2/5 was added to the treatment regimen (Reddy et al
., manuscript under review). In addition, the dramatic improvement was shown to arise from the immunomodulatory effects of BMT synergizing with the enzyme supplied by GT (Reddy et al
., manuscript under review). These are striking findings, considering that neonatal BMT alone provides no enzyme to the brain, and minimal clinical benefit (56
In the murine model of infantile neuronal ceroid lipofuscinosis (INCL; palitoyl protein thioesterase-1, PPT1−/−, deficiency), dramatic synergy is observed when CNS-directed AAV2/5 GT is combined with BMT. Untreated PPT−/− mice have a median lifespan of ~8 months. CNS-directed AAV2/5-mediated GT alone is modestly effective in this mouse model, extending the median lifespan to ~14 months. In contrast, BMT alone provides no clinical benefit. Remarkably, the median lifespan in animals receiving both BMT and CNS-directed AAV2/5 GT is ~17 months (Roberts et al., in preparation). In this same model, CNS-directed AAV2/5 in combination with a pharmacologic PPT1 mimetic (cystagon) provides no increase in the lifespan; however, combination-treated animals show improvement in motor function (Macauley et al., in preparation).
Although combination therapies are successful in several instances, they may sometimes show no improvement or actually worsen the disease. In the murine model of MPS-IIIB, the combination of CNS-directed AAV2/5 and BMT improved hearing and lysosomal inclusions, but the effects on motor function and lifespan were antagonistic when compared with either therapy alone (82
). In this case, the therapies may not provide enough correction to outweigh the deleterious effects of harsh conditioning regimens required for BMT. This study highlights the complex effects of BMT and other therapies on the disease. Similarly, in the murine model of GLD, when N
-acetyl cysteine (antioxidant) is combined with BMT, no positive effects on lifespan or behavioral performance were observed (Hawkins-Salsbury et al
., manuscript in preparation). This is surprising considering the dramatic increase in oxidative stress markers in GLD (83
It is not yet possible to predict which combinations will be successful. Undoubtedly, some pathogenic mechanisms are sufficiently down-stream of the primary insult that they will not be good therapeutic targets. A more complete understanding of the mechanisms of the disease in terms of the various pathways that are altered is needed. Also, a better understanding of the mechanism of action of various therapies, such as BMT, SRT or other small molecule drugs, could be helpful in designing more rational combination therapies. It is clear, however, that in certain instances therapies that show little or no efficacy when used alone, may add to or even synergize with other approaches. Therefore, treatments that have so far provided minimal efficacy in vivo (stop-codon read through, molecular chaperones and other small molecule drugs) may need to be revisited in the context of combination therapy. Given the complex nature of LSDs and the limitations of single therapies, it may be necessary to shift focus towards the development of combination treatments in order to more effectively treat these disorders. Ultimately, both single and combination therapies will be most effective if they are initiated before patients become symptomatic, highlighting the need for widespread newborn screening.
Conflict of Interest statement. None declared.