Generation of Lentiviral Vectors Encoding Engineered Glucocerebrosidases for Gene Therapy Treatment of Gaucher Disease

Megan Dunlevy and Salim El-Amouri, Ph.D., Dao Pan, Ph.D.

Introduction:
Gaucher disease is caused by a deficiency in glucocerebrosidase (GCase). This enzyme acts on the glycolipid glucocerebroside. When the enzyme is defective, glucosylceramide accumulates particularly in white blood cells, most often macrophages, spleen, liver kidneys, lungs, brain and bone marrow, resulting in organ malfunction, skeletal disorders, and severe neurologic complications. To this date, there is no treatment for the neurological complications is in Gauche patients. We have identified two peptides from receptor-binding domain of apolipoprotein E (E1 or E2) that can mediate protein transcytosis across the blood brain barrier (BBB) (Wang, D, et.al. 2013 PNAS). Fusing these two peptides with GCase will allow the enzyme in the circulating blood stream, after hematopoietic stem cells (HSCs) gene therapy, to pass through the BBB into the Central Nervous System (CNS) in order to treat the neurological complication associated with Gauche disease.

Concentration Method

Transduction
LV- IHK GC E1/ E2

GCaseE1

Spin at 4 degrees, 25,000 RPM for 1 hr.

Nucleus

Study Aim:
To generate GCase harboring lentiviral vectors (LV) that will be used for HSCs gene therapy in a mouse model of Gaucher disease.
C P P T C P P T I R E S I R E S

Add 20% sucrose to a new ultra-tube Tilt the tube and add virus

Secrete into Media/ bloodstream

Methods:

U5

GC E1

Lysosome

GFP

GCaseE2

LV- IHK GC E1 R U5 GC E2

GFP

LV- IHK GC E2 LV iHK GC E1/ E2

Spin at 4 degrees, 25,000 RPM for 1 hr.

p-Rev Transfection by Calcium Phosphate

293T Cells
GFP expression GCE1

Collect virus and store.

p-Env

Conclusion:
Lentiviral vectors co-expressing eGFP and GCase were generated by co-transfection of 293T cells using three helper plasmids. With successful transduction with LV-IHK GC E1/E2, Dami cells with the LV-IHK GC E1 could release into the media higher quantities of functional GCase than those with LV-iHK GC E2.

pDML GFP expression GCE2