**Problem:**
Cholesteatomas (CHSTs) are invasive lesions of the temporal bone,
skull base, and intracranial cavity primarily affecting the middle ear and can cause deafness, dizziness, facial nerve paralysis, brain abscess, meningitis, and death. CHSTs currently are treated only by a costly surgery associated with a high (15-70%) recurrence rate and risk of complications.
**Solution:**
The goal of the project is to develop a safely attenuated oncolytic herpes simplex virus (oHSV) that expresses therapeutic transgenes for enhanced effects, as a novel non-surgical and cost-effective therapy for CHSTs.
**Background:**
CHSTs are invasive growths of keratinizing squamous
epithelium in the middle ear and mastoid that cause significant morbidity including deafness, dizziness, facial paralysis, meningitis, and even death.
About 1 in 1,000 people with ear problems referred
to otolaryngology clinics has CHST. The annual incidence of CHST was reported as 3 per 100,000 in children
and 9.2 per 100,000 in adults with a male predominance of 1.4:1 (Barath et al.
2011). The late diagnosis of CHST is still a major cause of hearing loss in the
United States. Most of the time, patients do not know a CHST is present. The
expanding CHST generally causes destruction of the tympanic membrane (TM)
and ossicles (bones of hearing).
CHST is currently treatable only by a costly and complicated surgery associated with a 15-70% recurrence rate. Even in the US, CHST is a significant challenge for indigent patients and rural regions. The average charges for CHST surgical treatment, including personnel and facilities, is $40,000 USD. There are around 29,000 new CHST
diagnoses per year (given a base US population of 315 million people). This brings the total cost of CHST treatment, in the US alone, to more than one 1 billion dollars annually.
Depending on the type of procedure used, approximately 5-40% of CHST operations have residual CHST or 15% - 70% recurrence manifesting as early as 6 months or
many years later in the postoperative period (Choufani et al. 2007). CHSTs behave like tumors; unless the CHST is removed completely, it will regrow. Consequently, CHST patients need to have close follow-up care for life. The affected ear will be checked annually at the ENT clinic, and further surgeries are often required.
Nonsurgical medical managements (e.g., topical antibiotics, debridement, and systemic therapies) can be used to reduce the growth rate of CHST lesions. Unfortunately, the nonsurgical intervention does not remove the CHST and will not stop CHST growth or prevent further damage. Currently, CHSTs can only be removed by surgery; no drug will cure the disease.
Surgical complications: there is a risk of damaging other structures
(e.g., facial nerve, sigmoid sinus, temporal lobe and cerebellar dura). While CHST themselves can lead to serious complications such as permanent deafness and life-threatening illnesses such as meningitis, the surgeries can also cause neurovascular injury. Some individuals have medical conditions that make the risk of
surgical intervention unacceptably high. For example, the elderly can have diabetes or cardiopulmonary disease that places them at higher risk of perioperative complications.
Oncolytic virotherapy is emerging as a promising new therapeutic approach. Lytic viruses are safely attenuated but retain potency in proliferative cells and can be
endowed with transgenes for enhanced effects. Because of their lytic effects, the viruses have the potential to act as a “biologic surgery”. Compared to oncolytic virus treatment for malignant cancers, application of oHSV in CHST and other benign diseases treatment is currently unknown.
CHSTs development and destructive nature are dependent on expression of high levels of matrix metalloproteinases (MMPs). Here, we propose the design of a *ViruBot* (an oncolytic herpes simplex virus (oHSV) that expresses human tissue inhibitor of metalloproteinases 3 (TIMP3)). We proposed using this *ViruBot* to eradicate CHSTs via two parallel mechanisms:
1. cellular lytic effect of oncolytic virus; and
2. inhibition of MMPs activity via TIMP3.
**Goals:**
The goal for the current phase is to evaluate the cytotoxicity of *ViruBot* against primary CHST cells in vitro.
We anticipate that *ViruBot* will replicate efficiently in CHST cells and reduce the activity of MMPs, which will result in the destruction of CHST without affecting adjacent healthy epithelial cells. To validate the feasibility of this *ViruBot* as a novel approach for CHST treatment, two specific aims are proposed.
1. CHST tissues will be collected from ten patients during routine surgical resections and then subsequently cultured in the BSL-2 lab. The cultured CHST cells will be validated for characteristics of primary keratinocytes by K14/K10/P63 staining or RT-PCR. The expression of oHSV entry receptors in CHST
cells will also be tested by RT-PCR and flow cytometry. These primary human CHST cells will be used for Aim 2 (*ViruBot* testing), including three subdivided goals.
2. First, we propose to measure and quantify *ViruBot* cytotoxicity against CHST cells. A colorimetric MTS assay will be used to analyze the viability of CHST vs. control cells after *ViruBot* or control virus infection. Our preliminary data showed that our engineered ViruBot effectively kills primary CHST cells. More testing will need to be conducted and quantified in CHST cells from different patients (n=10) specimens.
Then, we propose to determine the replication efficiency of the *ViruBot* in CHST cells. The replication of the *ViruBot* will be quantified by the plaque
assay.
Finally, we propose to examine whether MMPs activities are inhibited in CHST cells by *ViruBot* infection. TIMP3 expression and MMPs activities will be quantified in CHST cells with or without *ViruBot* infection.
We expect this phase of the validation study will take approximatly six months following with preclinical in vivo study in Phase II, leading to subsequent clinical trials. Once validated, the *ViruBot* will provide the first non-surgical and cost-effective therapy for CHSTs.
