Cell and Gene Therapies for Mucopolysaccharidoses: Base Editing and Therapeutic Delivery to the CNS.

Although individually uncommon, rare diseases collectively account for a considerable proportion of disease impact worldwide. A group of rare genetic diseases called the mucopolysaccharidoses (MPSs) are characterized by accumulation of partially degraded glycosaminoglycans cellularly. MPS results in varied systemic symptoms and in some forms of the disease, neurodegeneration. Lack of treatment options for MPS with neurological involvement necessitates new avenues of therapeutic investigation. Cell and gene therapies provide putative alternatives and when coupled with genome editing technologies may provide long term or curative treatment. Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing technology and, more recently, advances in genome editing research, have allowed for the addition of base editors to the repertoire of CRISPR-based editing tools. The latest versions of base editors are highly efficient on-targeting deoxyribonucleic acid (DNA) editors. Here, we describe a number of putative guide ribonucleic acid (RNA) designs for precision correction of known causative mutations for 10 of the MPSs. In this review, we discuss advances in base editing technologies and current techniques for delivery of cell and gene therapies to the site of global degeneration in patients with severe neurological forms of MPS, the central nervous system, including ultrasound-mediated blood-brain barrier disruption.

A Prospective Treatment Option for Lysosomal Storage Diseases: CRISPR/Cas9 Gene Editing Technology for Mutation Correction in Induced Pluripotent Stem Cells.

Ease of design, relatively low cost and a multitude of gene-altering capabilities have all led to the adoption of the sophisticated and yet simple gene editing system: clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The CRISPR/Cas9 system holds promise for the correction of deleterious mutations by taking advantage of the homology directed repair pathway and by supplying a correction template to the affected patient's cells. Currently, this technique is being applied in vitro in human-induced pluripotent stem cells (iPSCs) to correct a variety of severe genetic diseases, but has not as of yet been used in iPSCs derived from patients affected with a lysosomal storage disease (LSD). If adopted into clinical practice, corrected iPSCs derived from cells that originate from the patient themselves could be used for therapeutic amelioration of LSD symptoms without the risks associated with allogeneic stem cell transplantation. CRISPR/Cas9 editing in a patient's cells would overcome the costly, lifelong process associated with currently available treatment methods, including enzyme replacement and substrate reduction therapies. In this review, the overall utility of the CRISPR/Cas9 gene editing technique for treatment of genetic diseases, the potential for the treatment of LSDs and methods currently employed to increase the efficiency of this re-engineered biological system will be discussed.

Identification of novel splice site mutation IVS9 + 1(G > A) and novel complex allele G355R/R359X in Type 1 Gaucher patients heterozygous for mutation N370S.

Gaucher disease is an autosomal recessive lysosomal storage disorder resulting from deficient glucocerebrosidase activity. More than 350 mutations that cause Gaucher disease have been described to date. Novel mutations can potentially provide insight into the glucocerebrosidase structure-function relationship and biochemical basis of the disease. Here, we report the identification of two novel mutations in two unrelated patients with type I (non-neuronopathic) Gaucher disease: 1) a splice site mutation IVS9 + 1G > A; and (2) a complex allele (cis) G355R/R359X. Both patients have a common N370S mutation in the other allele. The splice site mutation results from an intronic base substitution (G to A, c.1328 + 1, g.5005) at the donor splice site of exon and intron 9. The complex allele results from two point mutations in exon 8 of glucocerebrosidase (G to C at c.1180, g.4396, and T to C at c. 1192, g.4408) substituting glycine by arginine (G355R) and arginine by a premature termination (R359X), respectively. In order to demonstrate that G355R/R359X are in cis arrangement, PCR-amplified glucocerebrosidase exon 8 genomic DNA from the patient was cloned into the vector pJET1.2 in Escherichia coli TOP10® strain. Out of the 15 clones that were sequence analyzed, 10 contained the normal allele sequence and 5 contained the complex allele G355R/R359X sequence showing both mutations in cis arrangement. Restriction fragment length polymorphism analysis using Hph1 restriction endonuclease digest was established for the IVS9 + 1G > A mutation for confirmation and efficient identification of this mutation in future patients. Past literature suggests that mutations affecting splicing patterns of the glucocerebrosidase transcript as well as mutations in Gaucher complex alleles are detrimental to enzyme activity. However, compound heterozygosity with N370S, a mild mutation, will lead to a mild phenotype. The cases reported here support these past findings.

Two novel mutations in glucocerebrosidase, C23W and IVS7-1 G>A, identified in Type 1 Gaucher patients heterozygous for N370S.

Gaucher disease is an autosomal recessive lysosomal storage disorder resulting from deficient glucocerebrosidase activity. There have been nearly 300 mutations described to date. Novel mutations can potentially provide insight into the biochemical basis of the disease. Two novel mutations are described in two Type 1 Gaucher patients with N370S compound heterozygosity; a point mutation that causes an amino acid substitution at cysteine residue 23 for tryptophan, and a second point mutation within the splicing element at the 3' end of intron 7. Both mutations were identified by PCR amplification and sequence analysis of patient glucocerebrosidase genomic DNA. Restriction fragment length polymorphism analysis was established for both novel mutations for efficient identification in future patients. Past literature suggests that mutations affecting cysteine residues involved in disulfide bridges, as well as mutations affecting splicing patterns of the glucocerebrosidase transcript, are detrimental to enzyme activity. However, compound heterozygosity with N370S, a mild mutation, will lead to a mild phenotype. The cases reported here support these past findings.

Gaucher disease and the synucleinopathies: refining the relationship.

Gaucher disease (OMIM 230800, 230900, 231000), the most common lysosomal storage disorder, is due to a deficiency in the enzyme glucocerebrosidase. Gaucher patients display a wide spectrum of clinical presentation, with hepatosplenomegaly, haematological changes, and orthopaedic complications being the predominant symptoms. Gaucher disease is classified into three broad phenotypes based upon the presence or absence of neurological involvement: Type 1 (non-neuronopathic), Type 2 (acute neuronopathic), and Type 3 (subacute neuronopathic). Nearly 300 mutations have been identified in Gaucher patients, with the majority being missense mutations. Though studies of genotype-to-phenotype correlations have revealed significant heterogeneity, some consistent patterns have emerged to inform prognostic and therapeutic decisions. Recent research has highlighted a potential role for Gaucher disease in other comorbidities such as cancer and Parkinson's Disease. In this review, we will examine the potential relationship between Gaucher disease and the synucleinopathies, a group of neurodegenerative disorders characterized by the development of intracellular aggregates of α-synuclein. Possible mechanisms of interaction will be discussed.

Gaucher disease and cancer: concept and controversy.

Gaucher disease is an inherited disorder caused by a deficiency in the lysosomal hydrolase glucocerebrosidase. There is a wide spectrum of clinical presentations, with the most common features being hepatosplenomegaly, skeletal disease, and cytopenia. Gaucher disease has been classified into three broad phenotypes based upon the presence or absence of neurological involvement: Type 1 (nonneuronopathic), Type 2 (acute neuronopathic), and Type 3 (subacute neuronopathic). The two main treatment options include enzyme replacement therapy and substrate reduction therapy. Recently, discussion has escalated around the association of Gaucher disease and cancer, with conflicting reports as to whether Gaucher patients have an increased risk of malignancy. In this review, we present both the concept and controversy surrounding the association of Gaucher disease with cancer.

Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

Gaucher disease with prenatal onset and perinatal death due to compound heterozygosity for the missense R131C and null Rec Nci I GBA mutations.

Gaucher disease is an autosomal recessive disorder resulting from deficient activity of the lysosomal enzyme glucocerebrosidase (GBA, E.C.3.2.1.45). Three clinical forms of Gaucher disease have been described: type 1, nonneuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic (OMIM 230800, 230900, 231000). Over the past decade, recognition of a distinct, perinatal lethal form of Gaucher disease (PLGD) has led researchers and clinicians to evaluate Gaucher disease in the differential diagnosis of congenital ichthyosis and nonimmune hydrops fetalis. To date, more than 30 cases of PLGD have been genotyped and reported. It has been observed that homozygosity for recombinant GBA alleles, which are fundamentally null alleles, leads to early lethality, usually in utero or during the 1st few days of life, whereas genotypes involving a recombinant allele and a missense mutation may be less detrimental. Here, we report a case of Gaucher disease with prenatal onset and death within hours of birth, likely due to compound heterozygosity for the GBA Rec Nci I null allele and a R131C missense mutation. In view of the patient's severe clinical course, and based on reviews of other PLGD cases, we postulate that a missense mutation that abruptly disrupts the structure/function of GBA, in combination with a null allele, may result in early lethality in patients with PLGD. We also speculate that R131C is an extremely severe mutation that has occurred more than once in different populations and, in either the homozygous form or heterozygous with another severe mutation, will result in a poor prognosis.

Interaction of daunomycin with acetylated chromatin.

Anthracyclines are powerful chemotherapeutic agents for the treatment of many cancers. In many instances, they are currently used in combination with histone deacetylase inhibitors in order to enhance their efficiency. Not surprisingly and as part of their mode of action, these drugs interfere with gene expression, a process that has long been known to be mediated by histone acetylation. In this paper, we use analytical ultracentrifuge analysis, equilibrium dialysis, and circular dichroism to characterize the role of histone acetylation on the binding of antharcyclines to chromatin. We show that histone acetylation enhances the daunomycin-induced DNA dissociation from nucleosomes and decreases the extent of aggregation that results from the interaction in a way that is modulated by the presence or absence of linker histones. Histone acetylation increases the binding affinity of daunomycin by chromatin. Furthermore, the binding of anthracycline to acetylated chromatin sheds additional light into the conformational chromatin alterations resulting from core histone acetylation.

p53-based anti-cancer therapies: An empty promise?

Since its discovery in 1979, p53 has become the focus of intensive cancer-based research in laboratories around the world. The p53 protein mediates critical cellular functions including the response to genotoxic stress, differentiation, senescence, and apoptosis, and has been shown to be mutated in a large proportion of human cancers. These observations led many to speculate that targeting the p53 pathway would result in the development of successful anti-cancer treatments. In spite of this, 30 years later, p53 has yet to fulfill this promise. However, new insights into small molecule combination therapies, microRNA regulation, structuring of clinical trials, and potential involvement in stem cell regulation may help p53 reach its potential.

Gaucher disease among Chinese patients: review on genotype/phenotype correlation from 29 patients and identification of novel and rare alleles.

Gaucher disease, the most prevalent lysosomal storage disease, results from an inherited deficiency in the enzyme glucocerebrosidase. Three clinical forms of Gaucher disease have been described: Type 1 non-neuronopathic, Type 2 acute neuronopathic, and Type 3 subacute neuronopathic. Although Gaucher disease is panethnic, its presentation reveals some ethnic-specific characteristics. The Type 1 form is most common among Caucasian patients. In contrast, the majority of Chinese Gaucher disease patients have early age of onset, severe hematological and skeletal complications, and often neurological involvement, resulting in early childhood death. In this report, we review 29 cases of Gaucher disease from 23 unrelated patients and 6 patients from 3 non-consanguineous families. Among these patients, 13 were diagnosed as Type 1, 10 as Type 2, and 6 as Type 3. A novel mutation, del 205-209ACCTT, was identified in the heterozygous form with mutation R353W (c.1174C>T) by DNA sequence analysis in 2 Type 1 patients who are sibs. Mutation R353W was also found in the heterozygous form in 3 other Type 1 patients, with mutation L444P in 2 sibs and a second unknown Gaucher allele in the third patient. The Gaucher genotypes of the remaining Type 1 patients were F37V/L444P, G46E/L444P, R48W/R120W, N188S/L444P, Y205C/L444P, N370S/L444P, and L444P/unknown. It was noted that mutation N370S in the patient was linked to the pv1.1(-)(1) haplotype present in Jewish patients. Among the Type 2 patients, L444P was present in the heterozygous form with mutation F213I, L385P, or the complex allele (RecNci) in 5 patients. The second most common mutation, F213I, was found in the heterozygous form in 6 patients with mutations N382K, L383R, or L444P. The other mutations found in the Type 2 patients were P122L, V375L, Y363C, M416V, and 383-400del. The genotypes of the 6 Type 3 patients identified were D409H/D409H, D409H/G202R, G46E/N188S, N188S/unknown, and L444P/L444P. While D409H has been reported as being associated with cardiovascular/ocular involvements in Gaucher disease, there have been no such complications in these patients. As noted, the majority of the Gaucher mutations we identified in the Chinese patients were either rare or absent in other populations. With the exception of N370S and R353W found only in the Type 1 form, the majority of these mutations are severe ones that result in poor prognosis and often Types 2 and 3 Gaucher disease.

Generation of a conditional knockout of murine glucocerebrosidase: utility for the study of Gaucher disease.

Gaucher disease is a disorder of sphingolipid metabolism resulting from an inherited deficiency of the lysosomal hydrolase glucocerebrosidase. Affected individuals present with a spectrum of clinical symptoms ranging from hepatosplenomegaly, haematological abnormalities, and bone pain in type 1 disease, to severe neurodegeneration and premature death in types 2 and 3 disease. Although the basic biochemical defect is well characterized, there remains a poor understanding of the underlying pathophysiology of disease. In vitro studies suggest that macrophage glucocerebroside storage leads to tissue dysfunction through complex mechanisms involving altered intracellular calcium homeostasis and apoptosis. In order to study the pathogenic roles of these complex interactions, a viable animal model for Gaucher disease is needed. The complexity of this single gene disorder has been emphasized by the varied results of previous murine Gaucher models, ranging from perinatal lethality to phenotypically and biochemically asymptomatic animals. Recognizing the need to modulate the biochemical phenotype in mice to produce a relevant model, we have created a murine strain with key exons of the glucocerebrosidase gene flanked by loxP sites. We show that expression of Cre-recombinase in cells of hematopoietic and endothelial origin results in deficiency of glucocerebrosidase in the liver, spleen, bone marrow, and peripheral white cells. Glucocerebroside storage in this model leads to progressive splenomegaly with Gaucher cell infiltration and modest storage in the liver by 26 weeks of age. These results indicate the utility of this loxP GBA targeted murine strain for understanding the complex pathophysiology of Gaucher disease.

Expression of active alpha-N-acetylglucosaminidase/TAT Chimerae in cultured Spodoptera frugiperda cells.

We examined the production and secretion of fusion constructs containing alpha-N-acetylglucosaminidase, the enzyme deficient in Sanfilippo B, and either wildtype TAT or modified TAT in cultured Spodoptera frugiperda cells. All constructs exhibited successful expression of active enzyme, suggesting the future possibility of utilizing TAT/alpha-N-acetylglucosaminidase chimerae in enzyme replacement therapy.

Secretion of human glucocerebrosidase from stable transformed insect cells using native signal sequences.

The lysosomal hydrolase, glucocerebrosidase (GBA), catalyses the penultimate step in the breakdown of membrane glycosphingolipids. An inherited deficiency of this enzyme activity leads to the onset of Gaucher disease, the most common lysosomal storage disorder. Affected individuals range from adults with hepatosplenomegaly, haematological complications, and bone pain (type 1 disease) to children and neonates with severe neuronopathy leading to neurological degradation and premature death (type 2 and type 3 disease). Enzyme replacement therapy has become the standard of treatment for type I Gaucher disease but remains an expensive option, in part because of the cost of recombinant enzyme production using mammalian cell culture. Using a nonlytic integrative plasmid expression system, we have successfully produced active human GBA in stable transformed Sf9 (Spodoptera frugiperda) cells. Both the 39 and 19 amino acid native GBA signal sequences were capable of endoplasmic reticulum targeting, which led to secretion of the recombinant protein, although approximately 30% more enzyme was produced using the longer signal sequence. The secreted product was purified to apparent electrophoretic homogeneity using hydrophobic interaction chromatography and found to be produced in a fully glycosylated and a hypoglycosylated form, both of which cross-reacted with a human GBA-specific monoclonal antibody. The pH optimum (at pH 5.5) for activity of the recombinant enzyme was as expected for human GBA using the artificial substrate 4-methyl-umbelliferyl-beta-D-glycopyranoside. With initial nonoptimized expression levels estimated at 10-15 mg/L using small-scale batch cultures, stable transformed insect cells could provide a viable alternative system for the heterologous production of human GBA when grown under optimized perfusion culture conditions.

HIV TAT variants differentially influence the production of glucocerebrosidase in Sf9 cells.

Gaucher disease, the most common lysosomal storage disorder, is currently treated with enzyme replacement therapy. This approach, however, is ineffective in altering the progression of neurodegeneration in type 2 and type 3 patients due to the difficulty of transferring the recombinant enzyme across the blood-brain barrier. Human immunodeficiency virus type 1 trans-activating transcriptional activator protein (HIV TAT) contains a protein transduction domain that can be added to a fusion protein partner to allow for transport of the partner across membranes. Consequently, we examined the creation, production, and secretion of fusion constructs containing glucocerebrosidase and either wild-type TAT or modified TAT in Sf9 cells. All three constructs exhibited successful expression, with wild-type TAT chimeras showing lower levels of expression than modified TAT chimeras.

Glucocerebrosidase recombinant allele: molecular evolution of the glucocerebrosidase gene and pseudogene in primates.

Glucocerebrosidase is a lysosomal enzyme that hydrolyses the beta-glycosidic linkage of glucocerebroside, a ubiquitous sphingolipid present in the plasma membrane of mammalian cells. Deleterious mutations in the glucocerebrosidase gene result in Gaucher disease, the most prevalent lysosomal storage disease. Humans have one glucocerebrosidase functional gene and pseudogene that were located 16 kb apart on chromosome 1q21 and share 96% overall sequence similarity. Recombination between the two genes creates a 'complex allele' that renders glucocerebrosidase non-functional and accounts for >20% of the total Gaucher disease mutations in some population. The glucocerebrosidase pseudogene is absent in all other mammalian species surveyed so far. In order to learn more about the molecular evolution of the glucocerebrosidase functional gene and pseudogene, we have sequenced approximately 1.1 kb of the C-terminal region of these genes that encodes the enzyme catalytic site, from PCR-amplified genomic DNA of gorilla, chimpanzee, orangutan (the great apes), and squirrel monkey (a new-world monkey). In gorilla, chimpanzee, and orangutan, there are two copies of the glucocerebrosidase gene while the squirrel monkey possesses only a single copy. Similar to human, the second copy of glucocerebrosidase gene in gorilla and chimpanzee is non-functional because of a 55-bp deletion in exon 9, while that in orangutan appears to be unaffected and may still be functional. These data suggest that the glucocerebrosidase gene duplication event occurred after squirrel monkey divergence from the great apes, and that the exon 9 deletion that rendered the second copy of the glucocerebrosidase gene non-functional occurred prior to the divergence of gorilla and chimpanzee but after the divergence of orangutan from their common ancestor to human. The two genes in each species are least similar in gorilla and chimpanzee (97.8%) and most similar in orangutan (99.5%). None of the nucleotide variations in the GBA gene among the primates correspond to known mutations in Gaucher disease. Phylogenetic tree analysis using DNAstar and PAUP4.0 software indicates that gene conversion caused the evolution of glucocerebrosidase functional gene and pseudogene to be concerted.

Novel mutations in type 2 Gaucher disease in Chinese and their functional characterization by heterologous expression.

We investigated 10 unrelated Chinese patients with type 2 Gaucher disease and performed ex vivo expression for the novel mutations to characterize their functional defects. These patients were diagnosed by enzymatic assays and clinicopathologic features over the past five years in a national centre in China. Genomic DNA was sequenced by a two-stage PCR approach for mutations in the functional GBA gene. Novel mutations were expressed with baculovirus-transfected Sf21 cells. Six novel mutations were found (in traditional nomenclature): P122L, Y363C, N382K, L383R, L385P, and M416V. Review of reported mutations indicated clustering of type 2 mutations in three regions of the GBA gene. Expression of novel mutations revealed that the enzyme defect could arise from one of two mechanisms: loss of catalytic activity (Y363C and M416V) or enzyme instability (P122L and N382K).

RNA interference: past, present and future.

RNA interference (RNAi) is the sequence-specific gene silencing induced by double-stranded RNA. RNAi is mediated by 21-23 nucleotide small interfering RNAs (siRNAs) which are produced from long double-stranded RNAs by RNAse II-like enzyme Dicer. The resulting siRNAs are incorporated into a RNA-induced silencing complex (RISC) that targets and cleaves mRNA complementary to the siRNAs. Since its inception in 1998, RNAi has been demonstrated in organisms ranging from trypanosomes to nematodes to vertebrates. Potential uses already in progress include the examination of specific gene function in living systems, the development of anti-viral and anti-cancer therapies, and genome-wide screens. In this review, we discuss the landmark discoveries that established the contextual framework leading up to our current understanding of RNAi. We also provide an overview of current developments and future applications.

Knockdown of chimeric glucocerebrosidase by green fluorescent protein-directed small interfering RNA.

Gaucher disease, the most common type of lysosomal storage disorder, is characterized by an inherited deficiency of the membrane-associated hydrolase, glucocerebrosidase. Glucocerebrosidase catalyzes the hydrolysis of glucocerebroside to ceramide and glucose, a crucial step in the recycling of membrane sphingolipids. The exorbitant cost of the current treatment standard for Gaucher disease, enzyme replacement therapy, prevents many from receiving treatment. This limitation has led to a wide-spread search for more efficient and cost-effective methods of protein production and alternate therapies, resulting in a closer examination of glucocerebrosidase biosynthesis and current treatment techniques. The use of specific small interfering RNAs (siRNAs) to knock down target genes is an attractive option for studying such processes, though a glucocerebrosidase-specific siRNA has yet to be reported. We note, however, that green fluorescent protein (GFP)-directed siRNAs can not only provide a positive control to test siRNA delivery and system integrity, but also serve as a means to knock down a fusion partner without having to design siRNAs specific to the partner. After effectively co-transfecting COS-1 cells with enhanced GFP (EGFP)-tagged glucocerebrosidase constructs and GFP-directed siRNAs, we report successful knockdown of all EGFP-containing constructs at both the RNA and protein levels. This provides a method of examining enzyme biosynthesis and treatment options. Furthermore, this technique is applicable to other systems, since we have demonstrated the usefulness of GFP as a siRNA target in mammalian cells when fused to another gene of interest.