Screening of Chimeric GAA Variants in a Preclinical Study of Pompe Disease Results in Candidate Vector for Hematopoietic Stem Cell Gene Therapy

Pompe disease is a rare genetic neuromuscular disorder caused by acid alpha-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy (ERT) is the current standard of care, which prolongs the quality of life for Pompe patients. However, ERT has limitations due to lack of enzyme penetration into the central nervous system (CNS) and skeletal muscles, immunogenicity against the recombinant enzyme, and requires life-long biweekly infusions. In a preclinical mouse model, a clinically relevant promoter to drive lentiviral vector-mediated expression of engineered GAA in autologous hematopoietic stem and progenitor cells (HSPC) was tested with nine unique human chimeric GAA coding sequences incorporating distinct peptide tags and codon-optimization iterations. Vectors including glycosylation independent lysosomal targeting (GILT) tags resulted in effective GAA enzyme delivery into key disease tissues with enhanced reduction of glycogen, myofiber and CNS vacuolation, compared to non-tagged GAA in Gaa knockout mice, a model of Pompe disease. Genetically modified microglial cells in brains were detected at low levels, but provided robust correction. Furthermore, an aminoacid substitution in the tag added to reduced capacity to induce insulin signaling and there was no evidence of off-target effects. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model providing a promising vector candidate for further investigation.

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Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype

Blood ◽

10.1182/blood-2009-11-252874 ◽

2010 ◽

Vol 115 (26) ◽

pp. 5329-5337 ◽

Cited By ~ 50

Author(s):

Niek P. van Til ◽

Merel Stok ◽

Fatima S. F. Aerts Kaya ◽

Monique C. de Waard ◽

Elnaz Farahbakhshian ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Hematopoietic Stem Cells ◽

Pompe Disease ◽

Cell Function ◽

Lentiviral Vector ◽

Ex Vivo ◽

Genetically Engineered ◽

Hematopoietic Stem ◽

Enzyme Replacement

AbstractPompe disease (acid α-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector–mediated expression of human acid α-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid α-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid α-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

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Faculty Opinions recommendation of Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.739368425.793582178 ◽

2021 ◽

Author(s):

Frank Staal

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Lentiviral Vector ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Human Cd34

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882. Lentiviral Gene Therapy of Hematopoietic Stem Cells Results in Phenotype Correction in a Mouse Model of Pompe Disease

Molecular Therapy ◽

10.1016/s1525-0016(16)40285-6 ◽

2008 ◽

Vol 16 ◽

pp. S329

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Pompe Disease ◽

Hematopoietic Stem ◽

Lentiviral Gene Therapy

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Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model

Blood ◽

10.1182/blood-2010-04-278234 ◽

2010 ◽

Vol 116 (24) ◽

pp. 5130-5139 ◽

Cited By ~ 105

Author(s):

Ilaria Visigalli ◽

Stefania Delai ◽

Letterio S. Politi ◽

Carmela Di Domenico ◽

Federica Cerri ◽

...

Keyword(s):

Gene Therapy ◽

Mouse Model ◽

Hematopoietic Cell Transplantation ◽

Clinical Manifestations ◽

Cardiac Insufficiency ◽

Type I ◽

Hematopoietic Stem ◽

Enzyme Replacement ◽

Mps I ◽

Hsc Transplantation

AbstractType I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patients. Interestingly, therapeutic efficacy of HSC gene therapy is strictly dependent on the achievement of supranormal enzyme activity in the hematopoietic system of transplanted mice, which allows enzyme delivery to the brain and skeleton for disease correction. Overall, our data provide evidence of an efficacious treatment for MPS I Hurler patients, warranting future development toward clinical testing.

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Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector

Haematologica ◽

10.3324/haematol.2020.269142 ◽

2021 ◽

Author(s):

Yang Liu ◽

Maria Dahl ◽

Shubhranshu Debnath ◽

Michael Rothe ◽

Emma M. Smith ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Mouse Model ◽

Cord Blood ◽

Lentiviral Vector ◽

Bone Marrow Failure ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Clinical Gene Therapy ◽

Cord Blood Cells

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder with pure red blood cell aplasia associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1α short promoter, that can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19-deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice longterm and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

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Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidase

Science Translational Medicine ◽

10.1126/scitranslmed.aam6375 ◽

2017 ◽

Vol 9 (418) ◽

pp. eaam6375 ◽

Cited By ~ 39

Author(s):

Francesco Puzzo ◽

Pasqualina Colella ◽

Maria G. Biferi ◽

Deeksha Bali ◽

Nicole K. Paulk ◽

...

Keyword(s):

Pompe Disease ◽

Therapeutic Potential ◽

Scale Up ◽

Glycogen Storage Disease Type ◽

Neuromuscular Disorder ◽

The Body ◽

Respiratory Dysfunction ◽

Glycogen Accumulation ◽

Enzyme Replacement ◽

Hepatic Expression

Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa−/−) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa−/− mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector–mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.

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GLT1 gene delivery based on bone marrow-derived cells ameliorates motor function and survival in a mouse model of ALS

Scientific Reports ◽

10.1038/s41598-021-92285-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Natsuko Ohashi ◽

Tomoya Terashima ◽

Miwako Katagi ◽

Yuki Nakae ◽

Junko Okano ◽

...

Keyword(s):

Spinal Cord ◽

Gene Therapy ◽

Bone Marrow ◽

Mouse Model ◽

Lentiviral Vector ◽

White Blood Cells ◽

Glutamate Toxicity ◽

Pathological Lesion ◽

Arginase 1 ◽

Therapy Strategy

AbstractAmyotrophic lateral sclerosis (ALS) is an intractable neurodegenerative disease. CD68-positive bone marrow (BM)-derived cells (BMDCs) accumulate in the pathological lesion in the SOD1(G93A) ALS mouse model after BM transplantation (BMT). Therefore, we investigated whether BMDCs can be applied as gene carriers for cell-based gene therapy by employing the accumulation of BMDCs. In ALS mice, YFP reporter signals were observed in 12–14% of white blood cells (WBCs) and in the spinal cord via transplantation of BM after lentiviral vector (LV) infection. After confirmation of gene transduction by LV with the CD68 promoter in 4–7% of WBCs and in the spinal cord of ALS mice, BM cells were infected with LVs expressing glutamate transporter (GLT) 1 that protects neurons from glutamate toxicity, driven by the CD68 promoter, which were transplanted into ALS mice. The treated mice showed improvement of motor behaviors and prolonged survival. Additionally, interleukin (IL)-1β was significantly suppressed, and IL-4, arginase 1, and FIZZ were significantly increased in the mice. These results suggested that GLT1 expression by BMDCs improved the spinal cord environment. Therefore, our gene therapy strategy may be applied to treat neurodegenerative diseases such as ALS in which BMDCs accumulate in the pathological lesion by BMT.

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218. Gene Therapy and Enzyme Replacement in a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Molecular Therapy ◽

10.1016/j.ymthe.2006.08.243 ◽

2006 ◽

Vol 13 ◽

pp. S84

Author(s):

Michael Chang ◽

David Sleat ◽

Seng Cheng ◽

Marco Passini ◽

Peter Lobel ◽

...

Keyword(s):

Gene Therapy ◽

Mouse Model ◽

Neuronal Ceroid Lipofuscinosis ◽

Infantile Neuronal Ceroid Lipofuscinosis ◽

Enzyme Replacement ◽

Ceroid Lipofuscinosis

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Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

Virology Journal ◽

10.1186/s12985-021-01501-7 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Tugba Mehmetoglu-Gurbuz ◽

Rose Yeh ◽

Himanshu Garg ◽

Anjali Joshi

Keyword(s):

Gene Therapy ◽

Lentiviral Vector ◽

Suicide Gene ◽

Vector System ◽

Tat Protein ◽

Ccr5 Gene ◽

Hematopoietic Stem ◽

Therapy Strategy ◽

Hiv Tat ◽

A Cell

Abstract Background Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. Methods A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. Results Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. Conclusions Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

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