Qualifying Exam Presentation Proposal I: A Nonviral Gene Transfer - - PowerPoint PPT Presentation

▶

Feb 27, 2023 960 likes •1.16k views

Qualifying Exam Presentation Proposal I: A Nonviral Gene Transfer Agent Based on N-(carboxymethyl)- trans -4- hydroxyl-L-proline Sean O. Clancy Advisor: Aaron W. Harper 29 July 2002 Overview I. Background A. Types of vectors 1. Viral:

SLIDE 1

Qualifying Exam Presentation Proposal I: A Nonviral Gene Transfer Agent Based on N-(carboxymethyl)-trans-4- hydroxyl-L-proline

Sean O. Clancy Advisor: Aaron W. Harper 29 July 2002

SLIDE 2

Overview

I. Background
A. Types of vectors
1. Viral: retroviruses, adenoviruses, etc.
2. Nonviral: liposomes, peptides, polymers, etc.
B. Gene Delivery Process
C. Gene Delivery Requirements
II. N-(carboxymethyl)-trans-4-hydroxyl-L-proline based polymer
A. Reasoning
B. How it meets requirements
III. Synthesis
A. Monomer synthesis
B. Polymer synthesis
IV. Characterization and evaluation
A. NMR
B. Toxicity
C. Transfection efficiency

SLIDE 3

Types of Vectors

Viral: retroviruses, adenoviruses, etc.

– Must be de-evolved to be made safe. – Very expensive and sometimes dangerous. – Limits on size of DNA.

Nonviral: liposomes, peptides, polymers, etc.

– Toxicity. – Lack of targeting. – Ease of engineering.

SLIDE 4

Gene Delivery Process

DNA protection outside and inside cells.
Bypass or escape from endocytotic pathways.
Efficient release of DNA .
DNA delivery to most of the target cells.
At best:

– Efficient nuclear targeting. – High, persistent and adjustable therapeutic levels.

SLIDE 5

Gene Delivery Requirements

Be minimally toxic.
Efficiently transfect DNA.

– Balance toxicity vs. transfection efficiency.

Biodegradable ester linkages.
Tertiary amine groups in interior.
Primary amine groups on exterior.

SLIDE 6

N-(carboxymethyl)-trans-4-hydroxy-L-proline

Reason for choice

– 4-hydroxy-L-proline main component in collagen, which is nearly everywhere in the body of mammals.

How it will meet requirements

– Biodegradable ester linkages. – Internal tertiary amine to act as buffer.

N H HO CO2H

4-hydroxy-L-proline N-(carboxymethyl)-trans-4-hydroxy-L-proline

N HO CO2H CO2H

SLIDE 7

Monomer Synthesis

N H HO CO2H ClCH2CO2H NaOH N HO CO2

Na+

BaCl2 H2SO4 + BaSO4 CO2

Na+

N HO CO2

HO CO2H CO2H Ba2+ 1 2 3 4

SOCl2, MeOH N HO CO2CH3 CO2CH3 N HO CO2H CO2H 5

Chaouk, H.; Middleton, S.; Jackson, W. R.; Hearn, M. T. W.; International Journal of Bio- Chromatography; 1997, 2 (3), 153.

SLIDE 8

End Group Synthesis

H2N (CH2)3 HN (CH2)4 HN (CH2)3 NH2

Cl3CCH2OCOCl pyridine or aq. NaOH, r.t.

HN (CH2)3 N (CH2)4 HN (CH2)3 NH Troc Troc Troc

K2CO3 / DMF acetone, dry 70 deg C

HN (CH2)3 N (CH2)4 N (CH2)3 NH Troc Troc Troc Br OH HO

tosyl chloride, pyr., 0 deg C

HN (CH2)3 N (CH2)4 N (CH2)3 NH Troc Troc Troc O Tos

8 9 10 11

SLIDE 9

Polymer Synthesis

N O

O O

N O

O O

N O

O O

N O

O O O N O O O O N O O O O

O O N O O O O N

HO CO2CH3 CO2CH3

bulk polymerization under reduced pressure, 140 deg C

MeOH

5 6 7

SLIDE 10

Polymer Synthesis

N O

O O

N O

O O

N O

O O

N O

O O O N O O O O N O O O O H2N (CH2)3 HN (CH2)4 N (CH2)3 NH2 O NH2 (H2C)3 HN (CH2)4 N (CH2)3 NH2 O NH2 (H2C)3 NH (H2C)4 N (H2C)3 H2N O NH2 (H2C)3 NH (CH2)4 N (H2C)3 NH2 O NH2 (H2C)3 NH (H2C)4 N (H2C)3 NH2 O

HN (CH2)3 N (CH2)4 N (CH2)3 NH Troc Troc Troc O Tos

11 K2CO3, 18-C-6 acetone, dry reflux Polymer 7 Zinc, THF, H2O pH = 5.5 - 7.2 Troc cleavage

SLIDE 11

Characterization

1H and 13C NMR

– Determination of DB (degree of branching) by 13C

NMR with aid of model compounds.

– DB = (Nd + Nt)/(Nd + Nt + NlC + NlN) *

SEC with polystyrene and PAMAM as standards

N O

O O O O

N O

O O O O

N O

O O O O

N O

O O O O Terminal Dendritic Linear N Linear C

* Hawker, C. J.; Lee, R.; Frechet, J. M. J.; J. Am. Chem. Soc., 1991, 113, 4583.

SLIDE 12

Evaluation

Toxicity

– Measured using an MTT assay. – Pale yellow color of MTT (3-(4,5-cimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) turns to blue when cleaved by living cells.

Transfection efficiency

– Measure decrease or increase in target protein levels. – Include reporter gene in transfected DNA, such as green fluorescent protein (GFP).

SLIDE 13

Acknowledgements

The Harper Group

– Patrick J. Case – Jeremy C. Collette – Michael Julian – Cory G. Miller – Asanga B. Padmaperuma

Committee Members

Qualifying Exam Presentation Proposal I: A Nonviral Gene Transfer Agent Based on N-(carboxymethyl)-trans-4- hydroxyl-L-proline

Sean O. Clancy Advisor: Aaron W. Harper 29 July 2002

Overview

Types of Vectors

– Must be de-evolved to be made safe. – Very expensive and sometimes dangerous. – Limits on size of DNA.

– Toxicity. – Lack of targeting. – Ease of engineering.

Gene Delivery Process

– Efficient nuclear targeting. – High, persistent and adjustable therapeutic levels.

Gene Delivery Requirements

– Balance toxicity vs. transfection efficiency.

N-(carboxymethyl)-trans-4-hydroxy-L-proline

– 4-hydroxy-L-proline main component in collagen, which is nearly everywhere in the body of mammals.

– Biodegradable ester linkages. – Internal tertiary amine to act as buffer.

4-hydroxy-L-proline N-(carboxymethyl)-trans-4-hydroxy-L-proline

Monomer Synthesis

End Group Synthesis

Polymer Synthesis

Polymer Synthesis

Characterization

– Determination of DB (degree of branching) by 13C

NMR with aid of model compounds.

– DB = (Nd + Nt)/(Nd + Nt + NlC + NlN) *

Evaluation

– Measured using an MTT assay. – Pale yellow color of MTT (3-(4,5-cimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) turns to blue when cleaved by living cells.

– Measure decrease or increase in target protein levels. – Include reporter gene in transfected DNA, such as green fluorescent protein (GFP).

Acknowledgements

– Patrick J. Case – Jeremy C. Collette – Michael Julian – Cory G. Miller – Asanga B. Padmaperuma

– Robert Bau – Aaron W. Harper – G. K. Surya Prakash – William P. Weber – William H. Steier