Targeted enzyme delivery systems in lysosomal disorders: an innovative form of therapy for mucopolysaccharidosis.

Mucopolysaccharidoses (MPSs), which are inherited lysosomal storage disorders caused by the accumulation of undegraded glycosaminoglycans, can affect the central nervous system (CNS) and elicit cognitive and behavioral issues. Currently used enzyme replacement therapy methodologies often fail to adequately treat the manifestations of the disease in the CNS and other organs such as bone, cartilage, cornea, and heart. Targeted enzyme delivery systems (EDSs) can efficiently cross biological barriers such as blood-brain barrier and provide maximal therapeutic effects with minimal side effects, and hence, offer great clinical benefits over the currently used conventional enzyme replacement therapies. In this review, we provide comprehensive insights into MPSs and explore the clinical impacts of multimodal targeted EDSs.

Krabbe disease: One Hundred years from the bedside to the bench to the bedside.

This Review summarizes the progress in understanding the pathogenesis and treatment of Krabbe disease from the description of five patients in by Knud Krabbe until 2016. To determine the cause of this genetic disease, pathological and chemical analyses of tissues from the nervous systems of patients were performed. It was determined that these patients had a pathological feature known as globoid cell in the brain and that this consisted partially of galactosylceramide, a major sphingolipid component of myelin. The finding that these patients had a deficiency of galactocerebrosidase (GALC) activity opened the way to relatively simple diagnostic testing with easily obtainable tissue samples, studies leading to the purification of GALC, and cloning of the GALC cDNA and gene. The availability of the gene sequence led to the identification of mutations in patients and to the current studies involving the use of viral vectors containing the GALC cDNA to treat experimentally naturally occurring animal models, such as twitcher mice. Currently, treatment of presymptomatic human patients is limited to hematopoietic stem cell transplantation (HSCT). With recent studies showing successful treatment of animal models with a combination of HSCT and viral gene therapy, it is hoped that more effective treatments will soon be available for human patients. For this Review, it is not possible to reference all of the articles contributing to our current state of knowledge about this disease; however, we have chosen those that have influenced our studies by suggesting research paths to pursue. © 2016 Wiley Periodicals, Inc.

Long-term Improvements in Lifespan and Pathology in CNS and PNS After BMT Plus One Intravenous Injection of AAVrh10-GALC in Twitcher Mice.

Krabbe disease is an autosomal recessive disorder resulting from defects in the lysosomal enzyme galactocerebrosidase (GALC). GALC deficiency leads to severe neurological features. The only treatment for presymptomatic infantile patients and later-onset patients is hematopoietic stem cell transplantation (HSCT). This treatment is less than ideal with most patients eventually developing problems with gait and expressive language. Several naturally occurring animal models are available, including twitcher (twi) mice, which have been used for many treatment trials. Previous studies demonstrated that multiple injections of AAVrh10-GALC into the central nervous system (CNS) of neonatal twi mice resulted in significant improvements. Recently we showed that one i.v. injection of AAVrh10-GALC on PND10 resulted in normal GALC activity in the CNS and high activity in the peripheral nervous system (PNS). In the present study, a single i.v. injection of AAVrh10-GALC was given 1 day after bone marrow transplantation (BMT) on PND10. The mice show greatly extended lifespan and normal behavior with improved CNS and PNS findings. Since HSCT is the standard of care in human patients, adding this single i.v. injection of viral vector may greatly improve the treatment outcome.

Intravenous injection of AAVrh10-GALC after the neonatal period in twitcher mice results in significant expression in the central and peripheral nervous systems and improvement of clinical features.

Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessive disorder resulting from the defective lysosomal enzyme galactocerebrosidase (GALC). The lack of GALC enzyme leads to severe neurological symptoms. While most human patients are infants who do not survive beyond 2 years of age, older patients are also diagnosed. In addition to human patients, several naturally occurring animal models, including dog, mouse, and monkey, have also been identified. The mouse model of Krabbe disease, twitcher (twi) mouse has been used for many treatment trials including gene therapy. Using the combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of the adeno-associated virus serotype rh10 (AAVrh10) expressing mouse GALC in neonate twi mice we previously have demonstrated a significantly extended normal life and exhibition of normal behavior in treated mice. In spite of the prolonged healthy life of these treated mice and improved myelination, it is unlikely that using multiple injection sites for viral administration will be approved for treatment of human patients. In this study, we have explored the outcome of the single iv injection of viral vector at post-natal day 10 (PND10). This has resulted in increased GALC activity in the central nervous system (CNS) and high GALC activity in the peripheral nervous system (PNS). As we have shown previously, an iv injection of AAVrh10 at PND2 results in a small extension of life beyond the typical lifespan of the untreated twi mice (~40 days). In this study, we report that mice receiving a single iv injection at PND10 had no tremor and continued to gain weight until a few weeks before they died. On average, they lived 20-25 days longer than untreated mice. We anticipate that this strategy in combination with other therapeutic options may be beneficial and applicable to treatment of human patients.

Extended normal life after AAVrh10-mediated gene therapy in the mouse model of Krabbe disease.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). This deficiency results in accumulation of certain galactolipids including psychosine which is cytotoxic for myelin-producing cells. Treatment of human patients at this time is limited to hematopoietic stem cell transplantation (HSCT) that appears to slow the progression of the disease when performed in presymptomatic patients. In this study, adeno-associated virus (AAV) serotype rh10-(AAVrh10) expressing mouse GALC was used in treating twitcher (twi) mice, the mouse model of GLD. The combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of viral particles in neonate twi mice resulted in high GALC activity in brain and cerebellum and moderate to high GALC activity in spinal cord, sciatic nerve, and some peripheral organs. Successfully treated mice maintained their weight with no or very little twitching, living up to 8 months. The physical activities of the long-lived treated mice were comparable to wild type for most of their lives. Treated mice showed normal abilities to mate, to deliver pups, to nurse and to care for the newborns. This strategy alone or in combination with other therapeutic options may be applicable to treatment of human patients.

Krabbe disease: A personal perspective and hypothesis.

Introduction: Krabbe disease (KD) or globoid cell leukodystrophy (GLD) is one of the lysosomal disorders affecting central and peripheral nervous systems (CNS and PNS). It is caused by mutations on the galactocerebrosidase (GALC) gene. Affected individuals accumulate undegraded substrates and suffer from neuroinflammation. Methods: Hematopoietic stem cell transplantation (HSCT) has been partially successful in treating patients with KD when accomplished prior to the onset of symptoms. The success is credited to the ability of the hematopoietic stem cells in providing some GALC enzyme to the CNS and eradicating potential neuroinflammation. Combination of the HSCT with some other GALC-providing strategies has shown synergistic effects in the treatment of the mouse model of this disease. Results: Here, the possibility of eliminating HSCT in the treatment of human patients and replacing it with a single therapy that will provide sufficient GALC enzyme to the nervous systems is suggested. Such treatment, if started during the asymptomatic stage of the disease, not only may eradicate the enzyme deficiency, but may also keep any neuroinflammation at bay. Conclusion: Successful treatment of the KD may be possible by restoring consistent and sufficient GALC expression in CNS and PNS.

Can early treatment of twitcher mice with high dose AAVrh10-GALC eliminate the need for BMT?

Introduction: Krabbe disease (KD) is an autosomal recessive disorder caused by mutations in the galactocerebrosidase (GALC) gene resulting in neuro-inflammation and defective myelination in the central and peripheral nervous systems. Most infantile patients present with clinical features before six months of age and die before two years of age. The only treatment available for pre-symptomatic or mildly affected individuals is hematopoietic stem cell transplantation (HSCT). In the animal models, combining bone marrow transplantation (BMT) with gene therapy has shown the best results in disease outcome. In this study, we examine the outcome of gene therapy alone. Methods: Twitcher (twi) mice used in the study, have a W339X mutation in the GALC gene. Genotype identification of the mice was performed shortly after birth or post-natal day 1 (PND1), using polymerase chain reaction on the toe clips followed by restriction enzyme digestion and electrophoresis. Eight or nine-day-old affected mice were used for gene therapy treatment alone or combined with BMT. While iv injection of 4 × 1013 gc/kg of body weight of viral vector was used originally, different viral titers were also used without BMT to evaluate their outcomes. Results: When the standard viral dose was increased four- and ten-fold (4X and 10X) without BMT, the lifespans were increased significantly. Without BMT the affected mice were fertile, had the same weight and appearance as wild type mice and had normal strength and gait. The brains showed no staining for CD68, a marker for activated microglia/macrophages, and less astrogliosis than untreated twi mice. Conclusion: Our results demonstrate that, it may be possible to treat human KD patients with high dose AAVrh10 without blood stem cell transplantation which would eliminate the side effects of HSCT.

Advances in the Diagnosis and Treatment of Krabbe Disease.

Krabbe disease is an autosomal recessive leukodystrophy caused by pathogenic variants in the galactocerebrosidase (GALC) gene. GALC activity is needed for the lysosomal hydrolysis of galactosylceramide, an important component of myelin. While most patients are infants, older patients are also diagnosed. Starting in 1970, a diagnosis could be made by measuring GALC activity in leukocytes and cultured cells. After the purification of GALC in 1993, the cDNA and genes were cloned. Over 260 disease-causing variants as well as activity lowering benign variants have been identified. While some pathogenic variants can be considered "severe," others can be considered "mild." The combination of alleles determines the type of Krabbe disease a person will have. To identify patients earlier, newborn screening (NBS) has been implemented in several states. Low GALC activity in this screening test may indicate a diagnosis of Krabbe disease. Second tier testing as well as neuro-diagnostic studies may be required to identify those individuals needing immediate treatment. Treatment of pre-symptomatic or mildly symptomatic patients at this time is limited to hematopoietic stem cell transplantation. Treatment studies using the mouse and dog models have shown that combining bone marrow transplantation with intra-venous gene therapy provides the best outcomes in terms of survival, behavior, and preservation of normal myelination in the central and peripheral nervous systems. With earlier diagnosis of patients through newborn screening and advances in treatment, it is hoped that more patients will have a much better quality of life.

Enzyme replacement combinational therapy: effective treatments for mucopolysaccharidoses.

INTRODUCTION: Mucopolysaccharidoses (MPS), as a group of inherited lysosomal storage disorders (LSDs), are clinically heterogeneous and characterized by multi-systemic manifestations, such as skeletal abnormalities and neurological dysfunctions. The currently used enzyme replacement therapy (ERT) might be associated with several limitations including the low biodistribution of the enzymes into the main targets, immunological responses against foreign enzymes, and the high cost of the treatment procedure. Therefore, a suitable combination approach can be considered for the successful treatment of each type of MPS. AREAS COVERED: In this review, we provide comprehensive insights into the ERT-based combination therapies of MPS by reviewing the published literature on PubMed and Scopus. We also discuss the recent advancements in the treatment of MPS and bring up the hopes and hurdles in the futuristic treatment strategies. EXPERT OPINION: Given the complex pathophysiology of MPS and its involvement in different tissues, the ERT of MPS in combination with stem cell therapy or gene therapy is deemed to provide a personalized precision treatment modality with the highest therapeutic responses and minimal side effects. By the same token, new combinational approaches need to be evaluated by using drugs that target alternative and secondary pathological pathways.

Conditions for combining gene therapy with bone marrow transplantation in murine Krabbe disease.

Introduction: Krabbe disease (KD) is an autosomal recessive lysosomal disorder caused by mutations in the galactocerebrosidase (GALC) gene. This results in defective myelination in the peripheral and central nervous systems due to low GALC activity. Treatment at this time is limited to hematopoietic stem cell transplantation (HSCT) in pre-symptomatic individuals. While this treatment extends the lives of treated individuals, most have difficulty walking by the end of the first decade due to peripheral neuropathy. Studies in the murine model of KD, twitcher (twi) combining bone marrow transplantation (BMT) with AAVrh10-mGALC showed a great extension of life from 40 days to about 400 days, with some living a full life time. Methods: In order to find the optimum conditions for dosing and timing of this combined treatment, twi mice were injected with five doses of AAVrh10-mGALC at different times after BMT. Survival, as well as GALC expression were monitored along with studies of sciatic nerve myelination and possible liver pathology. Results: Dosing had a pronounced effect on survival and measured GALC activity. There was window of time after BMT to inject the viral vector and see similar results, however delaying both the BMT and the viral injection shortened the lifespans of the treated mice. Lowering the viral dose too much decreased the correction of the sciatic nerve myelination. There was no evidence for hepatic neoplasia. Conclusion: These studies provide the conditions optimum for successfully treating the murine model of KD. There is some flexibility in dosing and timing to obtain a satisfactory outcome. These studies are critical to the planning of a human trial combining the "standard of care", HSCT, with a single iv injection of AAVrh10-GALC.

BioImpacts: An emerging global journal.

The toddling BioImpacts has now grown into a young adult with strong opinions and perspectives, to a high-quality journal, and it has not been raised but by a family of professional editors, reviewers, authors, and even readers who had fantasized about a bright future and that fantasies are now coming true one-by-one.

Epitope-based vaccine design: a comprehensive overview of bioinformatics approaches.

Computational epitope-based vaccine design is the cornerstone of vaccine development. Owing to the selection of proper compositions [antigens (Ags), epitopes, peptide linkers, and intramolecular adjuvants], epitope-based vaccines are considered a cost- and time-effective approach resulting in the development of vaccines with maximal therapeutic efficacy and minimal adverse reactions. In this review, we provide insights into in silico epitope-based vaccine design and highlight vaccinology procedures used for the development of the next-generation vaccines with high effectiveness.

Bioengineered smart bacterial carriers for combinational targeted therapy of solid tumours.

Despite many endeavours for the development of new anticancer drugs, effective therapy of solid tumours remains a challenging issue. The current cancer chemotherapies may associate with two important limitations, including the lack/trivial specificity of treatment modalities towards diseased cells/tissues resulting in undesired side effects, and the emergence of drug-resistance mechanisms by tumour cells causing the failure of the treatment. Much attention, therefore, has currently been paid to develop smart and highly specific anticancer agents with maximal therapeutic impacts and minimal side effects. Among various strategies used to target cancer cells, bacteria-based cancer therapies (BCTs) have been validated as potential gene/drug delivery carriers, which can also be engineered to be used in diagnosis processes. They can be devised to selectively target the tumour microenvironment (TME), within which they may preferentially proliferate in the necrotic and anaerobic parts - often inaccessible to other therapeutics. BCTs are capable to sense and respond to the environmental signals, upon which they are considered as smart microrobots applicable in the controlled delivery of therapeutic agents to the TME. In this review, we aimed to provide comprehensive insights into the potentials of the bioengineered bacteria as smart and targeted bio-carriers and discuss their applications in cancer therapy.

Mathematical Models to Shed Light on Amyloid-Beta and Tau Protein Dependent Pathologies in Alzheimer's Disease.

The number of patients suffering from dementia due to Alzheimer's disease (AD) is constantly rising worldwide. This has accordingly resulted in huge burdens on the health systems and involved families. Lack of profound understanding of neural networking in normal brain and their interruption in AD makes the treatment of this neurodegenerative multifaceted disease a challenging issue. In recent years, mathematical and computational methods have paved the way towards a better understanding of the brain functional connectivity. Thus, much attention has been paid to this matter from both basic science researchers and clinicians with an interdisciplinary approach to determine what is not functioning properly in AD patients and how this malfunctioning can be addressed. In this review, a number of AD-related articles and well-studied pathophysiologic topics (e.g., amyloid-beta, neurofibrillary tangles, Ca2+ dysregulation, and synaptic plasticity alterations) has been literally surveyed from a computational and systems biology point of view. The neural networks were discussed from biological and mathematical point of views and their alterations in recent findings were further highlighted. Application of the graph theoretical analysis in the brain imaging was reviewed, depicting the relations between brain structure and function, without diving into mathematical details. Moreover, differential rate equations were briefly articulated, emphasizing the potential use of these equations in simplifying complex processes in relevance to pathologies of AD. Comprehensive insights were given into the AD progression from neural networks perspective, which may lead us towards potential strategies for early diagnosis and effective treatment of AD.

Significant correction of pathology in brains of twitcher mice following injection of genetically modified mouse neural progenitor cells.

Krabbe disease or globoid cell leukodystrophy is an autosomal recessive disorder resulting from mutations in the galactocerebrosidase (GALC) gene. These mutations lead to deficient GALC activity, storage of substrates of the enzyme, including psychosine, death to oligodendrocytes, decreased myelination, production of globoid cells and eventually death to the individual. While most affected individuals are infants, late-onset forms are also recognized. In addition to human patients, several animal models have been well characterized, including the twitcher mouse. A spontaneously transformed progenitor cell line was isolated from an astrocyte-enriched fraction of normal mice, partially characterized and transduced with a retrovirus-containing mouse GALC cDNA to produce increased GALC activity (20-30-fold above baseline). These cells, called MAR-52, were injected into the brains of newborn affected twitcher mice. While there was only a modest increase in lifespan and body weight, there was clear evidence for the correction of the astrocytic gliosis, normal appearing oligodendrocytes and evidence for remyelination. We demonstrate that the exogenously supplied neural progenitor cells can donate GALC enzyme to oligodendrocytes in the brains of affected mice resulting in normal myelination in the area of donor cells. At this time, hematopoietic stem cell transplantation provides the best outcome in affected mice and is the only treatment available for human patients, but it does not result in a cure even when performed in asymptomatic newborns. Complete correction probably will require a combined approach to effectively treat patients with Krabbe disease. With developments in the isolation and characterization of stem cells, this approach may improve the outcome for individuals diagnosed in the future.

Peptide-mediated drug delivery across the blood-brain barrier for targeting brain tumors.

INTRODUCTION: Transportation of the nutrients and other substances from the blood to the brain is selectively controlled by the brain capillary endothelial cells that form a restrictive barrier, so-called blood-brain barrier (BBB). Currently, there is no unimpeachable approach to overcome the BBB obstructiveness because the existing options are either invasive or ineffective. AREAS COVERED: This review delineates the biological impacts of BBB on brain drug delivery and targeting. The nanoscaled multifunctional shuttles armed with the targeting entities (e.g., antibodies and peptides) are discussed. Important insights are remarked into the combinatorial screening methodologies used for the identification of de novo peptides capable of crossing BBB and targeting the brain. EXPERT OPINION: Depending on the physicochemical properties of small molecules and macromolecules, they may cross the BBB and get into the brain either through passive diffusion or active/facilitated transportation and transcytosis in a very selectively controlled manner. Phage-derived shuttle peptides can specifically be selected against BBB endocytic machinery and used in engineering novel peptide-drug conjugates (PDCs). Nanoscaled multitargeting delivery systems encompassing PDCs can overcome the BBB obstructiveness and deliver drugs specifically to diseased cells in the brain with trivial side effects.

Enzyme replacement therapies: what is the best option?

Despite many beneficial outcomes of the conventional enzyme replacement therapy (ERT), several limitations such as the high-cost of the treatment and various inadvertent side effects including the occurrence of an immunological response against the infused enzyme and development of resistance to enzymes persist. These issues may limit the desired therapeutic outcomes of a majority of the lysosomal storage diseases (LSDs). Furthermore, the biodistribution of the recombinant enzymes into the target cells within the central nervous system (CNS), bone, cartilage, cornea, and heart still remain unresolved. All these shortcomings necessitate the development of more effective diagnosis and treatment modalities against LSDs. Taken all, maximizing the therapeutic response with minimal undesired side effects might be attainable by the development of targeted enzyme delivery systems (EDSs) as a promising alternative to the LSDs treatments, including different types of mucopolysaccharidoses ( MPSs ) as well as Fabry, Krabbe, Gaucher and Pompe diseases.

Debate on human aging and lifespan.

The issue of human lifespan has long been a matter of controversy among scientists. In spite of the recent claim by Dong et al that human lifespan is limited to 115 years, with the mounting improvements in biotechnology and scientific understanding of aging, we may be confident that aging will slow down over the course of the current century extending human longevity much longer than 115 years.

Gene therapy for CNS diseases - Krabbe disease.

This is a brief report of the 19th Annual Meeting of the American Society of Gene and Cell Therapy that took place from May 4th through May 7th, 2016 in Washington, DC, USA. While the meeting provided many symposiums, lectures, and scientific sessions this report mainly focuses on one of the sessions on the "Gene Therapy for central nervous system (CNS) Diseases" and specifically on the "Gene Therapy for the globoid cell leukodystrophy or Krabbe disease. Two presentations focused on this subject utilizing two animal models of this disease: mice and dog models. Different serotypes of adeno-associate viral vectors (AAV) alone or in combination with bone marrow transplantations were used in these research projects. The Meeting of the ASGCT reflected continuous growth in the fields of gene and cell therapy and brighter forecast for efficient treatment options for variety of human diseases.