Tag Archives: virus

ERV blogs on GMO Herpes vs severe cancer pain

As gene therapy emerges as one of the hottest areas of medical research, one thing that is striking is how it employs viruses – sometimes very nasty viruses – to deliver the gene to where it is needed in the human body.

Yesterday virologist Abbie Smith discussed another excellent example of this on the ERV blog in a post entitled “GMO Herpes vs. severs cancer pain”, describing how scientists at the Universities of Michigen and Pittsburgh have used a genetically modified herpes virus to deliver the preproenkephalin gene – which produced a precursor to pain-killing opiates – to the nerve cells of terminal cancer patients who were suffering from severe pain.

Abbie remarks that “This was one of the most depressing, yet hopeful, papers I have ever read.”. It’s difficult to disagree, after all most of the patients participating in the trial died within 3 months of it starting. But to focus on this sobering statistic would miss the reason for this study, namely that the pain-relief available to patients with severe chronic pain is often inadequate, as the drugs are not specific enough and cause unacceptable side effects when used at the high doses often required for prolonged periods of time. By targeting the opiate molecules to the nerve ccells themselves these side effects can be avoided, and more effective pain relief provided.

The paper “Gene Therapy for Pain: Results of a Phase I Clinical Trial” is available for anyone to read in PubMed Central and makes it very clear that this is a therapy that was discovered, evaluated and refined in animal models of different types of pain before entering this first clinical trial. The first two paragraphs of the introduction noting that:

A significant limitation to the development of analgesic drugs is that off-target effects at doses below the maximal analgesic threshold restrict the ability to selectively interrupt nociceptive neurotransmission1. To address this limitation, we developed a series of replication defective HSV-based vectors to deliver gene expression cassettes directly to DRG neurons from skin inoculation 2, 3. The anatomically defined projection of DRG axons allows targeting of specific ganglia by injection into selected dermatomes. In preclinical studies, the release of anti-nociceptive peptides or inhibitory neurotransmitters in spinal dorsal horn from the central terminals of transduced DRG neurons effectively reduced pain-related behaviors in rodent models of inflammatory pain, neuropathic pain, and pain caused by cancer4-9.

The human PENK gene encodes for preproenkephalin, a precursor protein proteolytically cleaved to produce the endogenous opioid peptides met- and leu-enkephalin. In the spinal cord, enkephalin peptides inhibit pain signaling through actions at presynaptic opioid receptors located on central terminals of primary afferent nociceptors and postsynaptic opioid receptors on second order neurons involved in nociceptive neurotransmission10. HSV vectors expressing opioid peptides appear to be particularly effective in animal models of inflammatory and cancer pain4, 5, 8.”

And in the conclusion:

In preclinical animal studies, skin inoculation of HSV vectors expressing PENK reduce acute hyperalgesic responses27, and reduce pain-related behaviors in models of arthritis28, formalin injection4, peripheral nerve damage6 and bone cancer5. Because this was the first human trial employing HSV vectors to achieve gene transfer, we elected to carry out the phase 1 clinical trial for safety and dose-finding in patients with pain caused by cancer…This Phase I clinical trial primarily addressed the question of whether intradermal delivery of NP2 to skin would prove to be safe and well tolerated by subjects. The small number of patients and the absence of placebo controls warrant circumspect interpretation of the secondary outcome measures. But the observation that subjects in the low dose cohort had little change in the NRS or SF-MPQ while subjects in the higher dose cohorts reported substantial reduction in NRS and improvement in SF-MPQ is encouraging.”

Encouraging is possibly an understatement, seeing clear evidence of therapeutic benefits in a Phase I trial like this is very promising, or as Abbie puts it “A trial turning out this successful is a great starting point for optimizing this kind of therapy.”.

Paul Browne

p.s. Those interested in a more detailed account of the research that led to this clinical trial can find it in this review published in 2008 and available to read online for free.

Mice pave the way to a cord blood transplant advance

Leukemia is a cancer of the blood or bone marrow that affects over 200,000 Americans and still kills thousands every year despite the great progress made over recent decades in developing  effective treatments for many leukemia types.  When undergoing treatment for leukemia many patients require hematopoietic stem cell (HSC) transplantation to replace the blood stem cells that are killed off along with the cancer cells by radiation or chemotherapy.  The most usual source of such HSCs is the blood or bone marrow of the patient themselves or a donor, but unfortunately it is often not possible to use the patient’s own cells because of the risk that some might be cancerous, while finding a donor whose cells are compatible with the patient is difficult, especially for members of ethnic minorities.  As a result of the growing numbers of patients waiting for suitable cells to become available for transplant scientists have turned to another source;  umbilical cord blood (CB) cells.  CB cells have the useful property of being partially immunologically privileged so that the match between the donor and recipient does not have to be as exact as for bone marrow or blood derived cells, but this advantage comes at the price that only small amounts of CB cells can be obtained from each umbilical cord.  To overcome this limitation on the use of CB cells scientists have sought to develop methods to expand in vitro the number of cells obtained from the umbilical cord before transplanting them into patients, and scientists recently announced the first successful clinical trial of in vitro expanded CB cells in leukemia patients (1) after a decade of research and refinement in mice.

Blood smear of the final blast crisis phase of chronic myelogenous leukemia, a disease whose treatment often includes hematopoietic stem cell transplant. Reproduced courtesy of the CDC Public Health Image Library.

Our story doesn’t however start with mice but with the fly Drosophila melanogaster,  a key model organism in developmental research.  Almost a century ago the geneticist Thomas Hunt Morgan identified a strain of D. Melanogaster which had characteristic notches in their wings, so the gene whose inactivation caused this trait was named Notch.  Since the revolution in molecular biology got underway in the 1980’s scientists studying D. melanogaster have learned that Notch  is a cell surface receptor which regulates the development of many tissues, and found that it plays a similar role in many other species, including mammals which have 4 versions of the Notch gene.  This all became relevant to the expansion of CB cells when a team lead by Dr. Irwin D. Bernstein of the Fred Hutchinson Cancer Research Center found that the Notch 1 gene was expressed in human hematopoietic  stem cells and decided to investigate the role of Notch 1 in regulating the ability of these cells to expand their numbers and subsequently differentiate into all the many different kinds of blood cell. A decade ago they used a modified virus to express Notch 1 in mouse bone marrow hematopoietic stem cells and found that these cells became immortal, producing far more cells than hematopoietic stem cells normally do in vitro, and that these cells could be made to differentiate into a wide range of blood cell types in vitro. However they found that while the Notch 1 expressing cells were incorporated into the bone marrow and gave rise to a wide variety of cell types when  transplanted along with unmodified hematopoietic stem cells into mice whose own bone marrow stem cells had been removed by radiation treatment , they did not do so when transplanted on their own (2).  This indicated that while using a viral vector to express Notch1 continually enhanced the ability of hematopoietic stem cells to self-renew and multiply it also impaired the other vital characteristic of these cells, namely their ability to differentiate into mature blood cells.

To overcome this problem Dr. Bernstein’s team turned to a modified version of Delta-1, the natural ligand of Notch, which could be used activate the Notch pathway and expand hematopoietic stem cells in vitro. When the cells were transplanted into the bone marrow the notch pathway would no longer be activated to such an extent and the cells could differentiate normally.  In the years that followed they tested and refined their Notch-mediated in vitro expansion technique, first using mouse bone marrow hematopoietic stem cells and when that was successful switching to human cord blood hematopoietic cells.  All this time they evaluated the ability of modified cells to engraft and repopulate blood and thymus with the full spectrum of blood cells by transplanting them into mice; including NOD-SCID mice which were genetically modified to be immunodeficient so that they could receive human cord blood cells that can then develop into a functioning immune system.

In the latest paper published online in Nature Medicine (1) Colleen Delaney and colleagues describe the final refinements to their technique. One major refinement concerned the source of the cell population to be expanded.  In previous studies they used a sub-population of CB  cells termed CD34+CD38- as the starting population in their in vitro expansion, a reasonable decision since CD34+CD38- are the most primitive form of hematopoietic cell with the greatest capacity to develop into the full range of other blood cell types.  Unfortunately the purification of CD34+ CD38- cells is a process that itself entails the loss of many CB hematopoietic cells, not an ideal situation when they are already in short supply.  So they compared a starting population of CD34+ cells with the more highly purified CD34+ CD38- population, and found that the CD34+ derived cells actually performed better when transplanted into mice.  The Notch-mediated in vitro expansion technique they had developed and refined over the preceding decade had produced cells that were able to engraft into the bone marrow a lot more quickly than untreated CB  hematopoietic stem cells and start producing immune cells earlier and in greater numbers,  it was now time to take it into human trials.

The phase I trials reported in the Nature Medicine paper involved transplanting the in vitro expanded CB cells alongside unmanipulated CB cells into high-risk leukemia patients, with the primary objective of evaluating the safety of the procedure.  At the end of the trial it was apparent that not only was the procedure not associated with any unexpected safety concerns, but that engraftment of the transplanted cells and production of immune cells was significantly enhanced, enabling the immune systems of the patients to recover more quickly.  While there are more and larger trials to come, this outcome raises the hope that umbilical cord blood cells will in future be able to offer many more leukemia patients the chance of earlier treatment and a quicker recovery.

Paul Browne Ph.D.

1)      Delaney C. et al. “Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution.” Nat. Med. Published online 17 January 2010 doi:10.1038/nm.2080

2)      Varnum-Finney B. Et al. “Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling” Nat. Med. Volume 6, Number 11, Pages 1278-1281 (2000) doi: 10.1038/81390