Molecular architecture determines brain delivery of a transferrin-receptor targeted lysosomal enzyme

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.

Cell-autonomous expression of the acid hydrolase galactocerebrosidase

Lysosomal storage diseases (LSDs) are typically caused by a deficiency in a soluble acid hydrolase and are characterized by the accumulation of undegraded substrates in the lysosome. Determining the role of specific cell types in the pathogenesis of LSDs is a major challenge due to the secretion and subsequent uptake of lysosomal hydrolases by adjacent cells, often referred to as “cross-correction.” Here we create and validate a conditional mouse model for cell-autonomous expression of galactocerebrosidase (GALC), the lysosomal enzyme deficient in Krabbe disease. We show that lysosomal membrane-tethered GALC (GALCLAMP1) retains enzyme activity, is able to cleave galactosylsphingosine, and is unable to cross-correct. Ubiquitous expression of GALCLAMP1 fully rescues the phenotype of the GALC-deficient mouse (Twitcher), and widespread deletion of GALCLAMP1 recapitulates the Twitcher phenotype. We demonstrate the utility of this model by deleting GALCLAMP1 specifically in myelinating Schwann cells in order to characterize the peripheral neuropathy seen in Krabbe disease.

Emerging Roles of Protein Deamidation in Innate Immune Signaling

Protein deamidation has been considered a nonenzymatic process associated with protein functional decay or “aging.” Recent studies implicate protein deamidation in regulating signal transduction in fundamental biological processes, such as innate immune responses. Work investigating gammaherpesviruses and bacterial pathogens indicates that microbial pathogens deploy deamidases or enzyme-deficient homologues (pseudoenzymes) to induce deamidation of key signaling components and evade host immune responses. Here, we review studies on protein deamidation in innate immune signaling and present several imminent questions concerning the roles of protein deamidation in infection and immunity.

High β-glucosidase (GBA) activity not attributable to GBA1 and GBA2 in live normal and enzyme-deficient fibroblasts may emphasise the role of additional GBAs

Beta-glucosidases (GBA) include GBA1, GBA2 and other β-glucosidases (non-GBA1-2). GBA1 is a lysosomal and GBA2 an extra-lysosomal enzyme. GBA1- and GBA2-deficient genetic conditions, with different phenotypes, are glucosylceramide (GC; the main GBA substrate) accumulating diseases. To study the activity profile of GBA, live fibroblasts were loaded with radioactive GC. The GC metabolism was measured in wild-type, GBA1-deficient (Gaucher disease) and GBA2-deficient (Gba2-/- mouse) cells. The differences found allowed the prediction of marked proportions of GBA1, GBA2, and particularly non-GBA1-2 (probably including GBA3, a cytosolic β-glucosidase) activity for wild-type cells. The high proportion of non-GBA1-2 suggests an important role of these enzymes.

G6PD DEFICIENCY

Context: Hyperbilirubinemia due to glucose 6 phosphate dehydrogenase(G6PD) deficiency can cause permanent neurological damage and death in neonates.Screening for the enzyme enables timely diagnosis and treatment in cases of G6PD relatedkernicterus. Knowledge of patient G6PD status is also important in treatment of malaria, adisease endemic in Pakistan. World Health Organization recommends mandatoryuse ofprimaquine for radical cure and eradication of malaria. Since, Primaquine, causes hemolysisin G6PD deficient cases, widespread adoption of the drug is viewed with caution. Aims: Thisstudy assessed frequency of G6PD deficiency in Pakistani noenates and examines the need forits screening based on local disease prevalence and malaria endemicity. Settings and Design:A cross sectional study was carried at Hematology Department, Army Medical College (NUST),in collaboration with Military Hospital, Rawalpindi, Pakistan.(January - August, 2011). Methodsand Material: The frequency of G6PD deficiency in newborn population was determined byquantitative (spectrophotometric) method. Cord blood (2.5 ml blood in K3EDTA bottle) sampleswere obtained from 240 newborns (male: female 1.2:1) after informed consent from parents.Statistical analysis used: Data obtained was analyzed using SPSS Windows version 17.Results: Frequency of G6PD deficient cases was 4.2%. Among the ten G6PD deficient patients,six had severe enzyme deficiency (<10% enzyme activity). Conclusions: The local prevalenceof G6PD deficiency and its potential complications qualify it as a disease that must be screenedfor. Also, prior knowledge of patient G6PD status enables the physician to revert to modifiedtreatment regimen for malaria only in enzyme deficient cases and not otherwise.

No excess of mitochondrial DNA deletions within muscle in progressive multiple sclerosis

Background: Mitochondrial dysfunction is an established feature of multiple sclerosis (MS). We recently described high levels of mitochondrial DNA (mtDNA) deletions within respiratory enzyme-deficient (lacking mitochondrial respiratory chain complex IV with intact complex II) neurons and choroid plexus epithelial cells in progressive MS. Objectives: The objective of this paper is to determine whether respiratory enzyme deficiency and mtDNA deletions in MS were in excess of age-related changes within muscle, which, like neurons, are post-mitotic cells that frequently harbour mtDNA deletions with ageing and in disease. Methods: In progressive MS cases ( n=17), known to harbour an excess of mtDNA deletions in the central nervous system (CNS), and controls ( n=15), we studied muscle (paraspinal) and explored mitochondria in single fibres. Histochemistry, immunohistochemistry, laser microdissection, real-time polymerase chain reaction (PCR), long-range PCR and sequencing were used to resolve the single muscle fibres. Results: The percentage of respiratory enzyme-deficient muscle fibres, mtDNA deletion level and percentage of muscle fibres harbouring high levels of mtDNA deletions were not significantly different in MS compared with controls. Conclusion: Our findings do not provide support to the existence of a diffuse mitochondrial abnormality involving multiple systems in MS. Understanding the cause(s) of the CNS mitochondrial dysfunction in progressive MS remains a research priority.

Platelets: An Efficient Depot for Generation and Distribution of Lysosomal Idua in Enzyme-Deficient MPS I Mice.

Abstract 3157 Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)-mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Platelets are terminally differentiated, enucleated cells full of secretory granules. Restricting transgene expression to platelets may reduce the risk for activating oncogenes in HSCs and most of their progeny, and take advantage of their rapid turnover and professional secretion function. In this study, we evaluated the feasibility of employing megakaryocyte-platelet as a depot for the over-expression and release of alpha-L-iduronidase (IDUA), the lysosomal enzyme deficient in patients with Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). Utilizing a human megakaryocytic DAMI cell line, we found that a hybrid human ankyrin-1 promoter (K) containing ALAS2 intron 8 enhancer and HS40 core element from human alpha LCR could introduce robust intracellular IDUA expression (over 500-fold of background) and significant enzyme release (120-fold). Upon introduction to megakaryocytic differentiation as verified by FACS analysis with PI staining and morphologic evaluation, the elevated intracellular IDUA levels remained unchanged, while released IDUA increased steadily by 30-fold. We then evaluated in vivo IDUA production/release from platelets in an enzyme-deficient MPS I mouse model. Lineage-negative bone marrow (BM) cells were isolated from MPS I mice using immunomagnetic cell sorting, and transduced twice with LV-K-IDUA-ires-GFP (KIiG) for a total multiplicity of infection at 30. Five-months after 1° transplantation, GFP+ platelets were detected by FACS analysis with CD41-PE staining and observed by Image X analysis. To generate animals with various transgene-dosages, 2° transplantation was performed into MPS I mice using a serial dilution of BM from 1° MPS/KIiG (51–65% transduction efficiency) with BM of MPS I. FACS analysis revealed that the percentage of GFP+ platelets was correlated directly to transduction efficiencies in 2° recipients as determined by real-time qPCR of BM 5 months post transplantation. Moreover, the intra-platelet IDUA enzyme activities were associated linearly with the changes of GFP+% platelets (r2=0.95) among MPS I mice with different transgene-dosages, suggesting that the lysosomal IDUA from transgene overexpression could be sorted and packaged into platelets with proper catalytic function. Noticeably 1% gene transfer efficiency as shown by 1% GFP+ platelets was sufficient to introduce the IDUA levels comparable to those found in wild-type platelets. To determine if platelet-derived IDUA could be released, we conducted Ca-induced platelet activation as verified by FACS analysis with Annexin V-APC and CD41-PE staining, resulting in linear correlation (r2=0.98) of released IDUA with intra-platelet IDUA. Interestingly, the plasma IDUA levels were also linearly correlated with GFP+% platelets (r2=0.96) among MPS I with different transgene-dosages. Competitive uptake assay using lymphoblastoid cells derived from a MPS I patient (LCLmps) demonstrated steady increase of intracellular IDUA with increasing amounts of extracellular enzyme levels, which was blocked by the presence of mannose-6-phosphate (M6P). This suggested that platelet-released IDUA retained its intercellular trafficking capability via M6P receptor. Finally normalization of aberrant lysosomal morphology was observed in LCLmps exposed to platelet-released IDUA as determined by in situ immunostaining with LysoTracker, indicating functional proficiency of platelet-derived IDUA to cross-correct deficit in patient's cells. These results demonstrate for the first time that megakaryocytes/platelets are capable of over-producing, packaging and storing a lysosomal enzyme which retains proper catalytic activity, lysosomal enzyme trafficking and endogenous M6PR-mediated uptake, as well as the ability of cross-correction in patient's cells. This data warrants further evaluation for the potential application of megakaryocytes/platelets in treating lysosomal storage diseases, especially for those, such as Gaucher disease, when the desirable enzyme is sensitive to neutral pH in serum and protection of enzyme in platelets may provide continuous, real-time enzyme release by low physiological levels of platelet activation. Disclosures: No relevant conflicts of interest to declare.