for importing nucleic acids, such as DNA, into the nuclei of osteoblast lineage cells (the bone forming cells of the skeleton)
Inventors
Strong, Donna D. • Linkhart, Thomas A. • Dean, David A.
Assignees
University of South Alabama • Loma Linda University • Northwestern University • US Department of Veterans Affairs
Publication Number
US-7741113-B2
Publication Date
2010-06-22
Expiration Date
2026-04-24
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Abstract
A plasmid, viral or linear DNA molecule containing a nucleic acid sequence derived from the promoter region of the hCol1α2 gene, which is selectively transported into the nuclei of cells in the osteoblast lineage. The sequence can be used independently as a nuclear entry sequence only, and/or as a nuclear entry sequence without regard to position, in a vector or linear DNA that directs gene expression and nuclear entry. The disclosure further includes a chimeric DNA sequence derived by the addition of osteoblast-specific enhancer sequences to the nuclear entry sequence/promoter sequence, to increase osteoblast-specific expression while retaining osteoblast-specific nuclear import. An enhancer sequence is derived from the promoter region of the human Core Binding Factor alpha 1 (Cbfa1/Runx2) gene. The Cbfa1/Runx2 promoter can be added to the sequence derived from, or alternatively, comprising the promoter region of the hCol1α2 gene. Also provided are methods of use of the novel sequences.
Core Innovation
The invention provides compositions and methods for importing nucleic acids, such as DNA, into the nuclei of osteoblast lineage cells by using an osteoblast-specific nuclear targeting sequence derived from the promoter region of the human type I alpha 2 procollagen (hCol1α2) gene. This nucleic acid sequence efficiently transports nucleic acids specifically into the osteoblast nucleus without requiring cell division and can be utilized independently or within vectors, plasmids, or linear DNA molecules to direct gene expression in osteoblasts.
The osteoblast-specific nuclear targeting sequence, exemplified by SEQ ID NO:1, comprises multiple binding sites for transcription factors uniquely expressed in osteoblast lineage cells, enabling selective nuclear import. The invention further includes chimeric sequences formed by combining the hCol1α2 nuclear entry/promoter sequence with osteoblast-specific enhancer sequences derived from the human Core Binding Factor alpha 1 (Cbfa1/Runx2) gene to enhance robust and specific transgene expression in osteoblasts while retaining nuclear targeting specificity.
Claims Coverage
The patent includes one independent claim covering the isolated or recombinant nucleic acid incorporating an osteoblast-specific nuclear targeting sequence with specified characteristics. The claim focuses on the sequence identity, contiguity with heterologous sequences, and its functional activity.
An isolated or recombinant nucleic acid including an osteoblast-specific nuclear targeting sequence
This nucleic acid comprises an expressible heterologous polynucleotide operably linked to a polynucleotide sequence that is at least 95% identical to SEQ ID NO:1 and is contiguous on either side with sequences not contiguous in the human genome. This osteoblast-specific nuclear targeting sequence mediates nuclear entry specifically into osteoblast lineage cells.
The independent claim covers nucleic acids featuring an osteoblast-specific nuclear targeting sequence defined by high sequence identity to SEQ ID NO:1, contiguous with heterologous sequences, operably linked to an expressible heterologous polynucleotide, and conferring osteoblast-specific nuclear entry. Dependent claims cover further aspects such as presence of promoters, enhancers, vectors, host cells, and kits incorporating such nucleic acids.
Stated Advantages
The osteoblast-specific nuclear targeting sequences provide highly efficient nuclear import of nucleic acids into non-dividing osteoblast lineage cells, achieving nuclear transformation efficiencies often exceeding 40-70%.
The invention enables specific targeting and expression of genes in osteoblast lineage cells, thereby restricting gene delivery and activity to skeletal tissues and improving safety and efficacy in gene therapy.
Combining the osteoblast-specific nuclear targeting sequence with osteoblast-specific enhancers enhances robust and specific transgene expression in osteoblasts, supporting therapeutic applications.
The nuclear targeting system allows for gene delivery and expression in non-dividing osteoblasts, overcoming a major barrier in gene therapy for bone-related diseases.
Documented Applications
Gene therapy for the treatment and prevention of bone diseases and disorders including osteoporosis, fracture repair, osteogenesis imperfecta, osteopetrosis, and abnormal bone growth conditions.
Development of transgenic and cellular models for studying bone metabolism and disease by expressing or repressing genes specifically in osteoblast lineage cells using the osteoblast-specific nuclear targeting sequence.
Methods for screening agents affecting osteoblast lineage cells by using cells harboring nucleic acids with the osteoblast-specific nuclear targeting sequence operably linked to reporters or genes that mimic bone disease states.
Targeted delivery and expression of therapeutic polypeptides or RNA molecules such as osteogenic factors, cytokines, transcription factors, and inhibitory RNAs to modulate bone growth, maintenance, repair, or degradation.
Localized gene deletion or activation in bone tissue using Cre recombinase under control of the osteoblast-specific nuclear targeting sequence to study gene function in vivo without extensive breeding of transgenic lines.
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