INTERNATIONAL RESEARCH NEWS:

Please note that most of the information we will publish on this page will probably be technical in nature. It will normally reflect the advances made in the name of the Spinal Cord Society and will increasingly emphasise advances made at the SCS Fort Collins Research facility. Be assured that we will make every effort to publish the very latest information irrespective of it's origin, but we may need your help from time to time. If you would like to make suggestions about our page, please email our project chairman at spinal@lionsclubtraralgon.asn.au or the site editor HERE.

SCS Laboratory - Fort Collins

A new Director of the Fort Collins laboratory has been appointed - Jesse Owens, who is himself a paraplegic and a research scientist of some years' standing. He has some innovative approaches to research and the lab has been expanded to a second floor, with further scientists being added, so this is a big step forward.


Genetic Engineering Unit Report


The main objectives of the genetic engineering work have been to create tools for expression of desired products to activate growth and form a permissive environment for regeneration in the central nervous system. This work has focused on creating DNA stable integration technology based on the patent SCS owns and the experimental work on animal model systems. The two main practical problems to accomplish this objective are to develop 1) a delivery system that involves getting the designed vector which carries the particular gene (cDNA) inside the cell, and 2) a stable integration system that involves inserting the gene into the host nucleus so it continues to express the gene product (protein). Our final goal is to apply these procedures to spinal cord injury. Since the last report, this project has dealt mainly with the stable integration system.

Our patented stable integration technology has established proof for the principle that genes (cDNA constructs) can be stably inserted into the host genome by using DNA recombination, replication and repair factors (REC/REP factors). The specific objectives for this project are two fold:

1) improve the efficiency of the insertion by using different combinations and concentrations of these factors on neuronal cells, and,

2) apply the technology to genes related to spinal cord injury treatments.

To determine the combinations of REC/REP factors, we use differentiated cells which model the invivo CNS. We obtained expression vectors for 15 different REC/REP factors. These vectors have been purified and tranfected into glial cells derived from neuronal progenitor cells. To determine the levels of stable integration into the recipient genome, we developed protocols for PCR detection of the inserted fragment. RT-PCR protocols are also being developed for determining the expression levels of the factors. Antibodies against the REC/REP factors were specifically made for the Society under a separate contract, and these antibodies are being used to assay for the expression levels in our cell models.

As the first step we analysed expression levels of the REC/REP factors on human neuronal progenitor cells when the cells were directed into the glial or neuronal path of differentiation. The possible candidates for DNA integration into non-dividing neuronal cells have also been found from these analyses. These are the factors, which are not significantly expressed in differentiated cells. Over-expression of these factors will be the cause of the DNA integration into non-dividing neuronal cells. We are moving forward In determining which combinations of these REC/REP factors give us the highest efficiency of stable inserted genes In differentiated neurons. Examining of expression levels of these factors in an adult nervous system is going on in parallel with the growth activation experiments. Once these expression levels have been established, and the most suitable combination has been tested in vitro, the in vivo work will begin.

The other project we are working on has been activation of neuronal growth. There are several reasons why adult neurons do not grow, but a fundamental problem is that injured adult neurons have lost their responsiveness to growth factors. Correlated with this is the lack of or very low number of receptors (Trk's) for the growth factors. Our earlier work has shown that it is possible to upregulate the expression of these receptors. Much in vitro work has been done characterizing combination treatments that make this possible. We are currently transferring these treatment protocols to spinal cord injury animal model. We have obtained the vector construct and RT-PCR primers for determining the upregulation of these receptors. When it is known that we have been successful on upregulating the receptors the treatment will be combined with stem cell bridging technology that is also concurrently being developed in the lab.

For the delivery system, we have focused on the SCS proprietary transfection systems (lipids and lipid-antibody mixes) with inconsistent results. These experiments are on hold waiting for new ideas from Drs. Holmberg and Owens.

Dr Aksel Soosar

SCS Laboratory, Fort Collins, CO

Glossary

mRNA-messenger - RiboNucleic Acid, template for syntheses of proteins.

PCR-Polymerase - Chain Reaction, a very sensitive method to determine known DNA sequences.

RT-PCR - Reverse Transcription-Polymerase Chain Reaction - modification of the PCR method which allows to determine low concentrations of mRNA.

Trk - certain type of receptors transferring signals from neurotrophic factors to cell nucleous.