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US-20260125678-A1 - DNA IMMORTALIZATION CONSTRUCT AND PERFORMING PRIMARY CELL IMMORTALIZATION

US20260125678A1US 20260125678 A1US20260125678 A1US 20260125678A1US-20260125678-A1

Abstract

A DNA immortalization construct includes a first nucleic acid sequence derived from the 5′ end of human CDKN2A gene exon 2. An EF1α promoter sequence is connected to the first sequence. A first LoxP sequence is connected to the EF1α promoter sequence. A human TERT gene sequence is connected to the first LoxP sequence. A second LoxP sequence is connected to the human TERT gene sequence. An internal ribosomal entrance site (IRES) sequence is connected to the second LoxP sequence. An antibiotic selection gene sequence is connected to the IRES sequence. An SV40 poly-A signal sequence is connected to the antibiotic selection gene sequence. A second nucleic acid sequence is connected to the SV40 poly-A signal sequence and derived from 3′ end of human CDKN2A gene exon 2.

Inventors

  • Zhiyong HE
  • Hua-Jun He
  • Kenneth D. Cole

Assignees

  • GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF COMMERCE

Dates

Publication Date
20260507
Application Date
20251125

Claims (20)

  1. 1 . A DNA immortalization construct ( 200 ) comprising: a first nucleic acid sequence ( 202 ) derived from the 5′ end of human CDKN2A gene exon 2; an EF1α promoter sequence ( 204 ) connected to the first sequence ( 202 ); a first LoxP sequence ( 206 ) connected to the EF1α promoter sequence ( 204 ); a human TERT gene sequence ( 208 ) connected to the first LoxP sequence ( 206 ); a second LoxP sequence ( 210 ) connected to the human TERT gene sequence ( 208 ); an internal ribosomal entrance site (IRES) sequence ( 212 ) connected to the second LoxP sequence ( 210 ); an antibiotic selection gene sequence ( 214 ) connected to the IRES sequence ( 212 ); an SV40 poly-A signal sequence ( 216 ) connected to the antibiotic selection gene sequence ( 214 ); and a second nucleic acid sequence ( 218 ) connected to the SV40 poly-A signal sequence ( 216 ) and derived from the 3′ end of human CDKN2A gene exon 2.
  2. 2 . The DNA immortalization construct ( 200 ) of claim 1 , wherein the first sequence ( 202 ) comprises 4.8 kilobases.
  3. 3 . The DNA immortalization construct ( 200 ) of claim 1 , wherein the antibiotic selection gene sequence ( 214 ) is a Zeocin resistance gene sequence.
  4. 4 . The DNA immortalization construct ( 200 ) of claim 1 , wherein the second sequence ( 218 ) comprises 2.1 kilobases.
  5. 5 . The DNA immortalization construct ( 200 ) of claim 1 , further comprising a vector backbone sequence ( 220 ) connected to the first sequence ( 202 ) and the second sequence ( 218 ).
  6. 6 . The DNA immortalization construct ( 200 ) of claim 5 , wherein the vector backbone sequence ( 220 ) is selected to promote homologous recombination.
  7. 7 . The DNA immortalization construct ( 200 ) of claim 1 , wherein the construct ( 200 ) is a synthetic plasmid.
  8. 8 . The DNA immortalization construct ( 200 ) of claim 1 , wherein the IRES sequence ( 212 ) is positioned between the human TERT gene sequence ( 208 ) and the antibiotic resistance gene sequence ( 214 ).
  9. 9 . The DNA immortalization construct ( 200 ) of claim 1 , wherein the first LoxP sequence ( 206 ) and the second LoxP sequence ( 210 ) allow removal of the human TERT gene sequence ( 208 ) by CRE-recombination.
  10. 10 . The DNA immortalization construct ( 200 ) of claim 1 , wherein the construct comprises the base sequence of SEQ ID NO: 9.
  11. 11 . A method ( 300 ) for immortalizing primary cells, the method comprising: providing a DNA immortalization construct ( 200 ) comprising a first nucleic acid sequence ( 202 ) derived from the 5′ end of a human CDKN2A gene exon 2; an EF1α promoter sequence ( 204 ) connected to the first sequence ( 202 ); a human TERT gene sequence ( 208 ) connected to the EF1α promoter sequence ( 204 ); an antibiotic resistance gene sequence ( 214 ); and a second nucleic acid sequence ( 218 ) derived from the 3′ end of the human CDKN2A gene exon 2; isolating primary cells ( 302 ) from human tissue; culturing the primary cells ( 302 ) to produce recipient cells ( 304 ); transfecting the recipient cells ( 304 ) with the DNA immortalization construct ( 200 ) using homologous recombination to produce transfected cells ( 306 ); culturing the transfected cells ( 306 ) in a medium ( 308 ) comprising an antibiotic ( 310 ) to select for transfected cells exhibiting antibiotic resistance; and isolating the transfected cells ( 306 ) exhibiting resistance to the antibiotic ( 310 ) to produce an immortalized cell line ( 312 ).
  12. 12 . The method of claim 11 , further comprising linearizing the DNA immortalization construct ( 200 ) prior to transfecting the recipient cells ( 304 ).
  13. 13 . The method of claim 11 , wherein linearizing comprises digesting the DNA immortalization construct ( 200 ) with a restriction enzyme.
  14. 14 . The method of claim 13 , wherein the restriction enzyme is PvuI.
  15. 15 . The method of claim 11 , wherein transfecting comprises electroporation.
  16. 16 . The method of claim 11 , further comprising: expanding the immortalized cell line ( 312 ); and cryopreserving the immortalized cell line ( 312 ).
  17. 17 . The method of claim 11 , wherein the primary cells ( 302 ) are CD8+ T-cells.
  18. 18 . The method of claim 11 , wherein the immortalized cell line ( 312 ) comprises a single copy of the human TERT gene sequence ( 208 ) inserted into the CDKN2A gene.
  19. 19 . The method of claim 18 , wherein the immortalized cell line ( 312 ) exhibits a decreased expression of the CDKN2A gene.
  20. 20 . The method of claim 11 , wherein the immortalized cell line ( 312 ) exhibits a normal karyotype and expresses cell surface markers consistent with the primary cells ( 302 ).

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/716,281 (filed Nov. 5, 2024), which is herein incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with United States Government support from the National Institute of Standards and Technology (NIST), an agency of the United States Department of Commerce. The Government has certain rights in this invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (21-066P1.xml; Size: 5200 bytes; and date of creation: Oct. 16, 2024) is herein incorporated by reference in its entirety. BACKGROUND The present invention generally relates to the field of genetic engineering and cell biology, and more particularly to techniques for immortalizing primary cells using a targeted DNA construct. The ability to culture and maintain viable cell lines in vitro is involved for a wide range of biological research and commercial applications, including drug discovery, vaccine development, and the production of biologics. Primary cells, harvested directly from living tissue, offer the advantage of reflecting the in vivo characteristics of their source, but they also present significant limitations. Primary cells typically have a finite replicative lifespan, undergoing senescence after a limited number of cell divisions. This Hayflick limit poses a significant obstacle to large-scale studies and commercial production, as it necessitates repeated harvesting of fresh tissue, introducing variability and increasing costs. Existing techniques for immortalizing cell lines, such as viral transduction or transfection with oncogenes like SV40 large T-antigen, often come at the cost of altering cellular function and genetic stability. Such immortalized cell lines may exhibit abnormal karyotypes, altered gene expression profiles, and even tumorigenic properties, raising concerns about their suitability as models for in vivo processes and their reliability in research and commercial applications. The resulting trade-off between replicative capacity and phenotypic fidelity presents an ongoing challenge for researchers and industry. Conventional approaches to cell immortalization often rely on introducing viral genes or activating endogenous oncogenes, effectively hijacking the cell's regulatory machinery to bypass normal growth control mechanisms. While these methods can achieve extended replicative lifespans, they often introduce uncontrolled genetic changes, creating cell lines that may deviate significantly from their primary cell counterparts. The random nature of viral integration and the pleiotropic effects of oncogenes can lead to unpredictable disruptions of cellular function, genetic instability, and even malignant transformation. Targeted approaches, such as introducing the catalytic subunit of telomerase (hTERT), offer a less disruptive alternative, but even these methods can be challenging to control, with variable expression levels and potential off-target effects. Furthermore, many current techniques rely on viral vectors for gene delivery, which raises additional safety concerns, especially for cell lines intended for therapeutic or clinical applications. The art therefore presents a spectrum of options, from highly disruptive but effective to more targeted but less reliable, with no single method offering both extended replicative capacity and preserved cellular function. Precise and controlled immortalization methods are especially useful for research areas requiring stable and representative cell models. Studies of cellular differentiation, tissue engineering, and drug discovery depend on cell lines that faithfully reflect the in vivo characteristics of their source. Moreover, the growing field of personalized medicine demands cell lines that can be derived from individual patients, while retaining their unique genetic and phenotypic profiles. Conventional immortalization techniques often fall short of these requirements, introducing variability and altering cellular functions in ways that can confound research results and limit the development of new therapies. The art thus involves a more refined approach, combining the ability to extend cellular lifespan with precise control over genetic manipulation, minimal disruption of cellular function, and a high degree of reproducibility. It is therefore an objective of the present invention to provide a method and a DNA construct for immortalizing primary cells by targeted insertion of a single copy of the hTERT gene, coupled with disruption of the CDKN2A gene, thereby overcoming the above-mentioned disadvantages of the prior art at least in part. Accordingly, methods and equipment for targeted gene insertion and homologous recombination enabling the production of stable, functionally representative immortalized cell lines would be advantageous and would be