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.

A Trimethoprim Conjugate of Thiomaltose Has Enhanced Antibacterial Efficacy In Vivo.

Trimethoprim is one of the most widely used antibiotics in the world. However, its efficacy is frequently limited by its poor water solubility and dose limiting toxicity. Prodrug strategies based on conjugation of oligosaccharides to trimethoprim have great potential for increasing the solubility of trimethoprim and lowering its toxicity, but they have been challenging to develop due to the sensitivity of trimethoprim to chemical modifications, and the rapid degradation of oligosaccharides in serum. In this report, we present a trimethoprim conjugate of maltodextrin termed TM-TMP, which increased the water solubility of trimethoprim by over 100 times, was stable to serum enzymes, and was active against urinary tract infections in mice. TM-TMP is composed of thiomaltose conjugated to trimethoprim, via a self-immolative disulfide linkage, and releases 4'-OH-trimethoprim (TMP-OH) after disulfide cleavage, which is a known metabolic product of trimethoprim and is as potent as trimethoprim. TM-TMP also contains a new maltodextrin targeting ligand composed of thiomaltose, which is stable to hydrolysis by serum amylases and therefore has the metabolic stability needed for in vivo use. TM-TMP has the potential to significantly improve the treatment of a wide number of infections given its high water solubility and the widespread use of trimethoprim.

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.

In vivo delivery of transcription factors with multifunctional oligonucleotides.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury.

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.

PET imaging of bacterial infections with fluorine-18-labeled maltohexaose.

A positron emission tomography (PET) tracer composed of (18)F-labeled maltohexaose (MH(18)F) can image bacteria in vivo with a sensitivity and specificity that are orders of magnitude higher than those of fluorodeoxyglucose ((18)FDG). MH(18)F can detect early-stage infections composed of as few as 10(5) E. coli colony-forming units (CFUs), and can identify drug resistance in bacteria in vivo. MH(18)F has the potential to improve the diagnosis of bacterial infections given its unique combination of high specificity and sensitivity for bacteria.

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.

Role of Pregabalin to Decrease Postoperative Pain in Microdiscectomy: A Randomized Clinical Trial.

Purpose The purpose of this study is to compare the effect of pregabalin in reducing the neuropathic pain in postoperative patients who have undergone single-level microdiscectomy for prolapsed intervertebral lumbar disc. Methods A randomized control clinical trial was conducted from June 2018 to April 2020 in three campuses Dr. Ziauddin University Hospital, Karachi, by two spinal surgeons. This study included 84 patients who underwent either emergency or elective microdiscectomy surgery. The patients randomized into two equal groups of 42, (group-A: pregabalin) and (group-B: placebo). Both groups also received routine analgesia along with the pregabalin and placebo capsules. In the intervention group, pregabalin was administered preoperative and postoperative defined times. The pain scores were recorded by visual analog scale (VAS) and Roland-Morris score system on the preoperative day and compared to the scores on follow-up on postoperative day seven. Results The pain scores were significantly better in group-A compared to group-B with similar baseline variables. The mean VAS scores of pains in group-A on postoperative day seven on follow-up were compared to VAS pain scores in group-B showing better pain control. The Roland-Morris scores were also significantly better for group-A. Conclusions The use of pregabalin in addition to the routine analgesia has better control of postoperative neuropathic pain in patients with single-level microdiscectomy compared to the patients who are receiving only routine analgesia. Other factors like cost, dose, side effects, and frequency should also be considered.

Persistent Shoulder Pain After Anterior Cervical Discectomy and Fusion (ACDF): Another Dual Pathology.

Purpose It is often difficult for the clinician to isolate the etiology of pain occurring either in the neck or shoulder because of the reason that neck pain can refer to the shoulder and vice versa. Concordance research has found that around one in 10 patients who were referred for cervical radiculopathy had comorbid shoulder pathology. The goal of this research is to analyze and correlate risk factors for persistent shoulder pain (non-dermatomal) following cervical spine surgery. Methods This was a single-center, retrospective study. The medical records of patients admitted for anterior cervical discectomy and fusion (ACDF) were reviewed from August 2018 to Feb 2021. Patients of both sexes and age more than 18 years who underwent ACDF (single/multiple levels) were included and the medical record was checked for whether they had persistent shoulder pain following ACDF. The proportion of patients undergoing shoulder surgery for associated rotator cuff tears and subacromial impingement were recorded. Results Seventy patients presenting with cervical prolapsed intervertebral disc (PID) were studied. A majority of our patients were females (n=48, 68.6%) and males (n=22, 31.4%) with an M:F ratio of 1:2 and the majority of patients were between the ages of 40 to 60 years (n=34, 48.6%). After surgical intervention (ACDF), 48 patients (68.6%) noted the cessation of shoulder symptoms (pain, weakness, and numbness) during their last visit. Rotator cuff tear (supraspinatus mainly) was the predominant finding in MRI in those who didn't improve after ACDF (n=18, 25.7%, p-value: 0.001). Twenty patients (28.6%) underwent acromioplasty and rotator cuff tendon repair and four patients responded well to subacromial injection. The C6-7 level was most commonly affected (n=48, 68.6%) followed by C5-6 level (n=19, 27.1%). No significant association was found between cervical levels with shoulder pathologies (p-0.171), though a significant association between a visual analog scale (VAS) score >7 after surgery with shoulder pathologies (p-0.001) was found. The C6-7 level was commonly affected in females (p=0.038) but no significant association between gender and shoulder pathologies was found (p=0.332). Conclusion Dual pathologies in patients with cervical PID are very common. It needs careful attention by doing thorough clinical examination and correlating patient symptoms with radiological investigations. A patient who presents with persistent shoulder pain after cervical spine surgery had a higher chance of having concurrent shoulder pathology, and they should be properly investigated and managed to alleviate the suffering of the patient.

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.

GO/PAMAM as a High Capacity Adsorbent for Removal of Alizarin Red S: Selective Separation of Dyes.

Adsorption of Alizarin Red S (ARS) on graphene oxide/poly(amidoamine) (GO/PAMAM) was studied at different ARS initial concentrations, temperatures, pHs, shaking rates and contact times. Adsorption sites of GO/PAMAM were phenolic -OHgroup of GO and amine groups (-NH2, -NH3+ and -NHR2+) of PAMAM dendrimer moieties of GO/PAMAM. At pH = 2 and 318 K, maximum adsorption capacity of the adsorbent was 1275.2 mg g-1 which is one of the highest capacity in the literature. Thus, GO/PAMAM in this work acted as a superadsorbent for ARS. At the incipient of adsorption, molecules were adsorbed on sites that was reaction-controlled step, (Ea = 114.5 kJ mol-1). Adsorption of on the remaining sites was diffusion-controlled. In alkaline media, two other types of ARS molecules were identified during that were adsorbed on and sites. Further increasing the pH of the solution, decreased the number these two sites and yielded a reduced adsorption capacity . Methylene blue (MB), thionine (Th), pyronin Y (PY), acridine orange (AO), methyl blue (MEB) and janus green (JG) dyes were selectively separated from their mixtures with ARS molecules using GO/PAMAM at pH of 2. The used adsorbent was recycled efficiently by using ethylenediamine very fast.

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.