The deadlocked jury was unable to reach a verdict after four days of deliberations in the case of 37-year-old Richard Nicoletti.
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On Friday, both the defense and prosecution agreed to proceed with 11 jurors instead of 12 after one was dismissed.
Two alternates also had medical issues preventing them from coming to court.
Philadelphia protesters gassed, maced on I-676 taking legal action
Nicoletti was charged with simple assault and reckless endangerment after he was caught on video pepper-spraying protesters on I-676 on June 1, 2020.
He was fired from the police force after the incident.
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In 2021, the charges were dismissed in court. The judge stated that the prosecution failed to prove Nicoletti's actions were criminal. That ruling was overturned and Nicoletti was brought to court for a second time.
READ | Charges refiled against former officer seen on video pepper-spraying protesters
Prosecutors argued that Nicoletti overstepped his position and put civilians at risk during the protest following George Floyd's murder.
However, Nicoletti's defense argued that he was following orders and clearing the highway before things got out of control.
"The prosecutor wouldn't be able to show to a fair-minded jury that Rich Nicoletti did anything other than his job that day," said defense attorney Fortunato Perri.
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FOP Lodge 5 President John McNesby released this statement on the mistrial:
"The double-standard of justice continues with DA Larry Krasner. He refuses to hold accountable the dozens of unlawful protesters and offenders who looted our great city, yet he works overtime to demonize a decorated, police officer for doing his job under very difficult conditions. The FOP and its members will continue its unwavering support of officer Nicoletti."
The district attorney's office declined to comment on the FOP's statement but did say it plans on retrying the case.
A hearing is scheduled for July 13.